Factor xi antibodies and methods of use

ABSTRACT

The present invention relates to monoclonal antibodies and antigen binding fragments thereof that bind to human Factor XI and activated Factor XI (“Factor XIa”), and pharmaceutical compositions and methods of treatment comprising the same.

This application claims the benefit of U.S. Provisional Application No.62/184,955 filed on Jun. 26, 2015 and U.S. Provisional Application No.62/341,568 filed on May 25, 2016, each of which is hereby incorporatedby reference in its entirety.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 23, 2016, isnamed “PAT056955-WO-PCT_SL.txt” and is 45,685 bytes in size.

BACKGROUND

Thrombosis refers to thrombus formation inside blood vessels, subsequentto a combination of hereditary and acquired risk factors, known asthrombophilia or hypercoagulable states. Vessel wall damage, stasis,increased platelets reactivity and activation of clotting factors aresome of the fundamental features of thrombosis. Thrombosis can occur inboth venous and arterial circulation and can result in the developmentof deep vein thrombosis (DVT), pulmonary embolism, and stroke. If athrombus occurs in the arterial system, down-stream ischemia can occur,leading to acute coronary syndromes (ACS), ischemic stroke, and acutelimb ischemia. Thrombus formation in the venous system typically leadsto deep venous thrombosis, pulmonary embolism and chronic thromboembolicpulmonary hypertension. Clots may also form in the left atrial appendagein patients with atrial fibrillation (AF), and dislodged thrombi mayresult in potentially devastating complications, i.e. thromboembolicstroke and systemic embolism. The currently available antithromboticmedications, including low molecular weight heparin (LMWH), thrombininhibitors, and Factor Xa (FXa) inhibitors, are all associated with asignificant risk of bleeding (Weitz J. I. (2010) Thromb. Haemost. 103,62). The development of an antithrombotic agent that does not affecthemostasis, and therefore does not result in bleeding complications,would be highly desirable.

Current anticoagulants are either injected or taken orally. Theinjectable anticoagulant LMWH is widely used and offers an improvedtherapeutic profile over formerly applied unfractionated heparin. Forthe past few decades the most commonly used oral anticoagulant has beenwarfarin. Warfarin has a narrow therapeutic window that requiresfrequent monitoring of the coagulation status, and shows a variety ofdrug-drug interactions. More recently, orally available direct FXa andthrombin inhibitors entered the anticoagulant market and areincreasingly applied.

LMWHs, FXa inhibitors, and thrombin inhibitors are all efficacious inthe prevention of post-operative venous thromboembolic disease, in thetreatment of spontaneous DVT and pulmonary embolism, and in the strokeprevention in atrial fibrillation. However, these anticoagulants arealso associated with bleeding complications that were generallycomparable to those observed with the older drugs warfarin andunfractionated heparin. In the ADVANCE-2 clinical trial, the FXainhibitor apixaban (Eliquis) was compared to the LMWH enoxaparin inpatients after total knee replacement. While acute apixaban therapy wasmore effective at preventing venous thromboembolic disease thanenoxaparin, both agents were associated with a significant risk ofbleeding. Clinically relevant bleeding occurred in 4% of patientsreceiving apixaban and in 5% of patients treated with enoxaparin(Lassen, M. R., et al. (2009) N. Engl. J. Med. 361, 594).

In the RE-LY trial, the direct thrombin inhibitor dabigatran (Pradaxa)was compared to warfarin in patients with atrial fibrillation and a riskof stroke (Connolly, S. J., et al. (2009) N. Engl. J. Med. 361, 1139).Chronic dabigatran therapy was associated with a significantly lowerrisk of stroke or systemic embolism. However, major bleedingcomplications occurred in 3.1% of patients receiving 150 mg per day ofdabigatran and in 3.4% of patients receiving warfarin (p=0.31).

Atrial fibrillation (AF) remains the most common cardiac arrhythmia inclinical practice, accounting for approximately one third ofhospitalizations for cardiac dysrhythmias. Currently, it is estimated toaffect more than 6 million patients in Europe and approximately 2.3million in the United States, and this number continues to grow rapidlybecause of the increasing proportion of the aging population. It isestimated that approximately 5% of the population over the age of 65years, and 10% of people aged over 80 years, will develop AF, however,the prevalence of AF is increasing beyond what is explained by agealone. AF risk factors such as hypertension, congestive heart failure,left ventricular hypertrophy, coronary artery disease and diabetesmellitus, and obstructive sleep apnea are also on the rise. As such, thenumber of affected individuals with AF is expected to increase two tothree times over the next three decades in western populations. (Kanneland Benjamin (2008) Med Clin North Am. 2008; 92:17-40; Bunch, et al.(2012) J Innovations of Card Rhythm Manag 2012; 3: 855-63).

The principal risk of AF is a four- to five fold increase in embolicstroke. The attributable risk for stroke associated with AF increasessteeply with age to 23.5% at ages 80 to 89. AF is associated with adoubling of mortality in both genders (Kannel and Benjamin 2008). AF isalso independently associated with cognitive decline and all forms ofdementia (Marzona, et al. (2012) CMAJ 2012; 184: 329-36; Geita et al2013; Bunch et al 2012).

Most patients with AF require life-long anticoagulation therapy toprevent cardioembolic stroke and systemic embolism. The CHA2DS2-VAScrisk score is a validated and widely used stratification tool to predictthromboembolic risk in atrial fibrillation patients and to identifypatients who should benefit from anticoagulation therapy (LIP 2011;Camm, et al. (2012) Eur Heart J 2012; 33: 2719-2747); the accumulatedevidence shows that CHA2DS2-VASc is at least as accurate as or possiblybetter than, scores such as CHADS2 in identifying patients who developstroke and thromboembolism and definitively better at identifying ‘trulylow-risk’ patients with AF. It is estimated that 85 to 90% of AFpatients will require anticoagulation therapy.

In a meta-analysis comprising 6 trials which evaluated the effect ofvitamin K antagonists (VKA) in reducing stroke and systemic embolism, ahighly significant risk reduction in stroke incidence (relative riskreduction of 67% for stoke) was observed. All-cause mortality wassignificantly reduced (26%) by adjusted-dose VKA vs. control (Hart,Pearce, and Aguilar (2007) Ann Intern Med 2007; 146:857-867). Aninternational normalized ratio (INR) target between 2 and 3 wasassociated with best benefit-risk ratio (Hylek et al (2003) N Engl JMed; 349:1019-1026) and universally adopted by international andnational guidelines.

In the recent years new oral anticoagulants (NOAC) also referred to asdirect oral anticoagulants (DOAC) have been approved and introduced toclinical practice. These drugs are at least as effective or even betterthan warfarin for reducing thrombo-embolic disease (Connolly, et al.(2009) N Engl J Med; 361:1139-51; Connolly, et al. (2011) N Engl J Med;364:806-17; Patel, et al. (2011) N Engl J Med 2011; 365:883-91). NOACwere also associated with large reductions in the most devastatingcomplications of warfarin namely hemorrhagic stroke and intracranialhemorrhage. Major bleeding events were similar or slightly lower thanwell conducted warfarin therapy. In addition NOAC are associated with alower potential for drug-drug interaction than warfarin and could beused without routine monitoring; this is expected to ease their use ineveryday medical practice.

Despite recent improvements, bleeding risk continues to be high with theuse of anticoagulants. For instance, the annual incidence of major andclinically relevant non major bleeding was 14.9% and the annualincidence of major bleeding events was 3.6% in patients treated withrivaroxaban in the ROCKET study (Patel et al 2011). The annual incidenceof major bleeding was >5% in patients at a high risk for bleedingdefined as HAS Bled risk score ≥3 (Gallego, et al. (2012) Caro ArrhythmElectrophysiol.; 5:312-318). Major bleeding is a particularly relevantclinical outcome; for instance in the ROCKET study, once major bleedinghas occurred, all-cause mortality rate was 20.4% in the rivaroxabangroup and 26.1% in the warfarin group. Once major bleeding events haveoccurred stroke and systemic embolism occurred in 4.7% and 5.4% ofpatients in rivaroxaban and warfarin groups, respectively (Piccini, etal. (2014) Eur Heart J; 35:1873-80). Hospital stay, transfusion of bloodproducts and resources utilization were also severely impacted by theoccurrence of major bleeding. Bleeding risk is also a major reason fornot receiving anticoagulants in eligible patients. In the Euro HeartSurvey on Atrial Fibrillation comprising data from 182 hospitals in 35countries and 5333 ambulant and hospitalized AF patients, only 67% ofeligible patients received oral anticoagulant at discharge (Nieuwlaat,et al (2005) Eur Heart J; 26, 2422-2434).

A high unmet medical need therefore exists for a safer therapy which canreduce AF thromboembolic complications such as stroke, systemicembolism, cognitive decline and mortality with comparable efficacy asexisting therapy but with a lower bleeding liability.

SUMMARY

The present invention relates to monoclonal antibodies binding to humancoagulation Factor XI and XIa (activated Factor XI) (hereinafter,sometimes referred to as “FXI”, “FXIa,” and similar terms), andpharmaceutical compositions comprising the same and methods of treatmentcomprising administering the same. The development of an anti-thromboticagent that is efficacious in the prevention and treatment of thrombosisor thromboembolic disease/disorder (e.g., thrombic stroke, atrialfibrillation, stroke prevention in atrial fibrillation (SPAF), deep veinthrombosis, venous thromboembolism, pulmonary embolism, acute coronarysyndromes (ACS), ischemic stroke, acute limb ischemia, chronicthromboembolic pulmonary hypertension, systemic embolism) but carries noor only minimal bleeding risk would meet a sizable unmet medical need.

In specific aspects, antibodies (e.g., human, chimeric, humanizedmonoclonal antibodies) provided herein bind with similarly high affinityto the catalytic domain (CD) of human FXIa and FXI and induces aninactive protease domain conformation in FXIa.

The isolated anti-FXI and/or anti-FXIa antibodies described herein,e.g., the full IgGs described herein with two binding sites, bind FXIand/or FXIa with an equilibrium dissociation constant (K₀) of less thanor equal to 100 pM. For example, the isolated antibodies describedherein may bind to human FXI and/or FXIa with a K_(D) of less than orequal to 100 pM, less than or equal to 50 pM, less than or equal to 45pM, less than or equal to 40 pM, less than or equal to 35 pM, less thanor equal to 20 pM, or less than or equal to 10 pM. More specifically,the isolated antibodies described herein may also bind human FXI and/orFXIa with a K_(D) of less than or equal to 34 pM, as measured by surfaceplasmon resonance (SPR), e.g., BIACORE™ assay, or less than or equal to4 pM, as measured by solution equilibrium titration assay (SET); and mayalso bind cynomolgus monkey FXI and/or FXIa with a K_(D) of less than orequal to 53 pM, as measured by BIACORE™ assay, or less than or equal to4 pM, as measured by SET. In specific aspects, isolated antibodiesdescribed herein (e.g., NOV1401) bind human FXI and FXIa with anapparent K_(D) of less than or equal to approximately 5 pM (e.g., 4.7pM) and 2 pM (e.g., 1.3 pM), respectively, for example as measured bysolution equilibrium titration assay (SET). In specific embodiments,anti-FXI/FXIa antibodies described herein bind to cynomolgus monkeyFXI/FXIa with an apparent K_(D) of approximately 12.5 (±6.6) pM for FXIaand approximately 5.0 (±0.7) pM as measured by SET (see, e.g., Example2). In specific embodiments, anti-FXI/FXIa antibodies described hereinbind rabbit FXI and/or FXIa with a K_(D) of approximately 20 (±2) nM. Inspecific aspects, anti-FXI/FXIa antibodies described herein bind human,cynomolgus monkey and rabbit FXI and/or FXIa, but do not specificallybind mouse or rat FXI.

The isolated anti-FXI and/or anti-FXIa antigen binding fragmentsdescribed herein, e.g., Fab fragments and other fragments containing onebinding site, bind FXI and/or FXIa, with an equilibrium dissociationconstant (K₀) of less than or equal to 10 nM. For example, the isolatedantigen binding fragments described herein may bind to human FXI and/orFXIa with a KD of less than or equal to 10 nM, less than or equal to 5nM, less than or equal to 1 nM, less than or equal to 500 pM, less thanor equal to 305 pM, less or equal to 62 pM. More specifically, theisolated antigen binding fragments described herein may also bind humanFXI and/or FXIa with a KD of less than or equal to 305 pM.

The present invention relates to an isolated antibody, or antigenbinding fragments thereof, that binds to human, rabbit, and cynomolgusmonkey FXIa. The invention also relates to an isolated antibody, orantigen binding fragments thereof, that binds within the catalyticdomain of FXI and/or FXIa, specifically to the surface of the activesite region.

The present invention also relates to an isolated antibody, or antigenbinding fragments thereof, that binds FXI and/or FXIa and furthercompetes for binding with an antibody as described in Table 1 (e.g.,NOV1401). As described here, “competition” between antibodies and/orantigen binding fragments thereof signifies that both antibodies (orbinding fragments thereof) bind to the same, or overlapping, FXI and/orFXIa epitope (e.g., as determined by a competitive binding assay, by anyof the methods well known to those of skill in the art). As used herein,an antibody or antigen binding fragment thereof does not “compete” withan FXI and/or FXIa antibody or antigen binding fragment of the invention(e.g., NOV1401 or NOV1090) unless said competing antibody or antigenbinding fragment thereof binds the same FXI and/or FXIa epitope, or anoverlapping FXI and/or FXIa epitope, as an antibody or antigen bindingfragment of the invention. As used herein, a competing antibody orantigen binding fragment thereof does not include one which (i)sterically blocks an antibody or antigen binding fragment of theinvention from binding its target (e.g., if said competing antibodybinds to a nearby, non-overlapping FXI and/or FXIa epitope andphysically prevents an antibody or antigen binding fragment of theinvention from binding its target); and/or (ii) binds to a different,non-overlapping FXI and/or FXIa epitope and induces a conformationalchange to the FXI and/or FXIa protein such that said protein can nolonger be bound by an FXI and/or FXIa antibody or antigen bindingfragment of the invention in a way that would occur absent saidconformational change.

In one embodiment, isolated antibodies, or antigen binding fragmentsthereof, bind FXI and/or FXIa and further compete for binding with anantibody as described in Table 1 bind to a majority of the amino acidsof the epitope(s) bound by said antibody of Table 1. In anotherembodiment, isolated antibodies, or antigen binding fragments thereof,that bind FXI and/or FXIa and further compete for binding with anantibody as described in Table 1 bind to all of the epitope(s) bound bysaid antibody of Table 1.

In one embodiment, isolated antibodies, or antigen binding fragmentsthereof, bind to active FXI (FXIa) and leads upon binding to the activeFXI (FXIa) catalytic domain to FXIa changing its conformation to aninactive conformation. In another embodiment, said isolated antibodiesor antigen binding fragments thereof further induce a change in whichthe N-terminal 4 residues, loops 145, 188 and 220 of said inactiveconformation are shifted and/or disordered compared to the activeconformation.

In one embodiment, isolated antibodies, or antigen binding fragmentsthereof, bind to FXI (e.g., human FXI) and upon binding to FXI preventthe FXI catalytic domain from assuming an active conformation, in whichloops 145, 188 and 220 are ordered as in the structure of the FXIacatalytic domain.

In one embodiment, isolated antibodies, or antigen binding fragmentsthereof, bind to FXI and upon binding to FXI prevents the FXI catalyticdomain from assuming an active conformation, in which the N-terminal 4residues, loops 145, 188 and 220 are ordered as in the structure of theFXIa catalytic domain.

In one embodiment, isolated antibodies, or antigen binding fragmentsthereof, bind to FXI and upon binding to FXI prevents the FXI catalyticdomain from assuming an active conformation by inducing conformationalchanges in the zymogen structure, further leading to an inhibited FXIconformation closely related to that observed when binding to FXIa.

In one embodiment, isolated antibodies, or antigen binding fragmentsthereof, bind to FXI and/or FXIa and upon binding to FXI and/or FXIa andforming an antibody: antigen complex with the catalytic domain of FXIand/or FXIa cause a shift and/or disorientation of loops 145, 188 and220 when compared with the uncomplexed structure of the catalytic domainof active Factor XI (FXIa).

In one embodiment, isolated antibodies, or antigen binding fragmentsthereof, bind to FXI and/or FXIa upon binding to FXI and/or FXIa andforming an antibody: antigen complex with the catalytic domain of FXIand/or FXIa causes a shift and/or disorientation of the N-terminal 4residues, loops 145, 188 and 220 when compared with the uncomplexedstructure of the catalytic domain of active Factor XI (FXIa).

In one embodiment, isolated antibodies, or antigen binding fragmentsthereof, bind to active FXI (FXIa) and cause the FXI (FXIa) catalyticdomain to change its conformation to an inactive conformation, in whichloops 145, 188 and 220 are shifted and/or disoriented compared to theactive conformation.

In one embodiment, isolated antibodies, or antigen binding fragmentsthereof, bind to FXI and prevent the catalytic domain from assuming anactive conformation by inducing a conformational changes in the zymogenstructure, thereby leading to an inhibited FXI conformation closelyrelated to that observed when binding to FXIa.

The present invention also further relates to an isolated antibody, orantigen binding fragments thereof, that binds the same epitope as anantibody as described in Table 1 (e.g., NOV1401).

The binding affinity of isolated antibodies and antigen bindingfragments described herein can be determined by solution equilibriumtitration (SET). Methods for SET are known in the art and are describedin further detail below. Alternatively, binding affinity of the isolatedantibodies, or fragments, described herein can be determined by surfaceplasmon resonance measurements, e.g., in BIACORE™ assays. Methods forBIACORE™ kinetic assays are known in the art and are described infurther detail below.

The isolated anti-FXI and/or FXIa antibodies and antigen bindingfragments described herein can be used to inhibit the direct or indirectactivation of Factor IX (also known as FIX), Factor X (FX), and/orthrombin, and/or the binding to platelet receptors, and thereby canprevent activation of the intrinsic and/or common coagulation pathways.

The isolated anti-FXI and/or FXIa antibodies and antigen bindingfragments described herein can be used to inhibit the direct or indirectactivation of Factor IX (also known as FIX), Factor X (FX), and/orthrombin with an IC₅₀ of less than or equal to 100 nM, less than orequal to 50 nM, less than or equal to 35 nM, less than or equal to 25nM, less than or equal to 10 nM, or less than or equal to 5.2 nM. Morespecifically, an isolated antibody or antigen binding fragments thereofas described herein can inhibit the direct or indirect activation ofFactor IX (also known as FIX), Factor X (FX), and/or thrombin with anIC₅₀ of less than or equal to 100 nM, less than or equal to 50 nM, lessthan or equal to 35 nM, less than or equal to 25 nM, less than or equalto 10 nM, or less than or equal to 5.2 nM. More specifically, anisolated antibody or antigen binding fragments thereof as describedherein can inhibit the direct or indirect activation of Factor IX (alsoknown as FIX), Factor X (FX), and/or thrombin with an IC₅₀ of less thanor equal to 100 nM, less than or equal to 50 nM, less than or equal to35 nM, less than or equal to 25 nM, less than or equal to 20 nM, or lessthan or equal to 18 nM. More specifically, an isolated antibody orantigen binding fragments thereof as described herein can inhibit thedirect or indirect activation of Factor IX (also known as FIX), Factor X(FX), and/or thrombin with an IC₅₀ of less than or equal to 100 nM, lessthan or equal to 50 nM, less than or equal to 35 nM, less than or equalto 25 nM, less than or equal to 10 nM, or less than or equal to 5 nM. Ina specific embodiment, an anti-FXI/FXIa antibody described herein, orantigen binding fragment thereof, inhibits FXIa-mediated activation ofits native substrate FIX with an IC₅₀ of less than or equal to 2 nM,e.g., 1.8 nM.

The isolated anti-FXI and/or anti-FXIa antibodies, or antigen bindingfragments thereof, may be used to inhibit (e.g., block the activationof) the intrinsic and/or common coagulation pathways, e.g., viainhibiting FXI and/or FXIa-mediated activation of FIX. The isolatedanti-FXI/FXIa antibodies, or antigen binding fragments thereof, maytherefore be used to prevent clotting or the propagation of clotting.The isolated antibodies, or antigen binding fragments thereof, may beused to prevent, treat, or ameliorate such coagulation disorders as deepvein thrombosis and stroke (e.g., ischemic stroke) by inhibitingFXI-mediated activation of FIX.

In specific embodiments, anti-FXI and/or anti-FXIa antibodies, orantigen binding fragments thereof, are capable of prolonging theclotting time (e.g., time until a blood clot starts to form) of humanplasma in a concentration-dependent manner as determined by an aPTTassay, for example as described in the Examples Section. In a specificembodiment, clotting time (aPTT) was doubled compared to baseline at atotal anti-FXI antibody (e.g., NOV1401) concentration in the range of 10nM to 20 nM, for example approximately 14 nM or 15 nM, as determined byan aPTT assay. In particular embodiments, anti-FXI and/or anti-FXIaantibodies, or antigen binding fragments thereof, are capable ofprolonging the clotting time of human plasma in aconcentration-dependent manner with an IC50 in the range of 5 nM to 20nM, for example approximately 13 nM, as determined by the aPTT assay,for example as described in the Examples Section.

In specific embodiments, anti-FXI and/or anti-FXIa antibodies describedherein, or antigen binding fragments thereof, are capable of prolongingthe clotting time (e.g., time until a blood clot starts to form) ofhuman plasma by at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, or 2 fold, e.g., in aconcentration-dependent manner, as determined by an aPTT assay, forexample as described in the Examples Section. In specific embodiments,anti-FXI and/or anti-FXIa antibodies described herein, or antigenbinding fragments thereof, are capable of prolonging the clotting time(e.g., time until a blood clot starts to form) of human plasma by atleast 1.4 fold, 1.5 fold, 1.6 fold, or 1.7 fold, as determined by anaPTT assay, for example as described in the Examples Section.

In specific aspects, anti-FXI and/or anti-FXIa antibodies, or antigenbinding fragments thereof, described herein is capable of reducing theamount of thrombin, in a concentration-dependent manner, in a thrombingeneration assay (TGA) in human plasma, which measures the effect ofFXIa inhibition on the thrombing→FXIa feed-forward loop in the presenceof very low tissue factor (TF) concentrations. In particularembodiments, anti-FXI and/or anti-FXIa antibodies, or antigen bindingfragments thereof, described herein is capable of reducing the amount ofthrombin in a thrombin generation assay (TGA) in human plasma with anIC₅₀ value in the range of 10 nM to 30 nM, for example approximately 20nM or 24 nM, and a residual thrombin concentration of approximately 159nM.

In specific aspects, provided herein are antibodies (e.g., antibodies inTable 1 such as NOV1401 or antibodies comprising the HCDRs 1-3 and LCDRs1-3 of NOV1401), or antigen binding fragments thereof, whichspecifically binds to the catalytic domain of human FXI and/or FXIa, andwhich has a terminal elimination half-life (t %) of total antibodies incynomolgus monkeys as approximately 14-15 days. In specific embodiments,such anti-FXI/FXIa antibodies exhibit an absolute subcutaneous (s.c.)bioavailability of approximately 61-66%.

In a specific embodiment, an antibody or antigen binding fragmentthereof provided herein (e.g., antibody described in Table 1, such asNOV1401), which specifically binds to human FXI and/or FXIa, exhibitsone or more (e.g., two, or three, or four, or five, or six, or seven),or all, of the following characteristics:

-   -   (i) specifically binds to a catalytic domain (CD) of human FXI        and FXIa, for example, with an apparent K_(D) of approximately        1-2 pM and 4-5 pM respectively;    -   (ii) prolongs clotting time as evaluated by activated partial        thromboplastin time (aPTT) assay;    -   (iii) inhibits thrombin generation in human plasma through        inhibition of FXI activation by activated factor XII (FXIIa) and        by thrombin, respectively;    -   (iv) shows antithrombotic and anticoagulant activity in FXI−/−        mice reconstituted with human FXI;    -   (v) reduces or prolongs the reduction of free FXI (FXI_(f))        levels, for example, in cynomolgus monkeys;    -   (vi) has a terminal elimination half-life of total antibody of        approximately 14-15 days, for example, in cynomolgus monkeys;    -   (vii) specifically binds to human and monkey FXI and/or FXIa but        does not specifically bind to mouse or rat FXI and/or FXIa; and    -   (viii) contacts one or more (e.g., two, three, four, five, six,        or seven, or more), or some, or all, of the following residues        of human FXI (Swissprot numbering): Pro410, Arg413, Leu415,        Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472-Glu476,        Tyr521-Lys527, Arg548, His552, Ser575, Ser594-Glu597, and        Arg602-Arg604.

The isolated anti-FXI and/or FXIa antibodies, or antigen bindingfragments thereof, as described herein can be monoclonal antibodies,human or humanized antibodies, chimeric antibodies, single chainantibodies, Fab fragments, Fv fragments, F(ab′)2 fragments, or scFvfragments, and/or IgG isotypes (e.g., IgG1 such as human IgG1). Inspecific embodiments, anti-FXI and/or anti-FXIa antibodies describedherein are recombinant human antibodies. In specific embodiments,anti-FXI and/or anti-FXIa antibodies described herein are humanIgG1/lambda (A) antibodies. In specific embodiments, anti-FXI and/oranti-FXIa antibodies described herein are human IgG1/lambda (λ)antibodies comprising an Fc domain engineered to reduce the potentialfor effector function (e.g., ADCC and/or CDC), for example a human Fcdomain comprising D265A and/or P329A substitutions.

The isolated anti-FXI and/or FXIa antibodies, or antigen bindingfragments thereof, as described herein can also include a framework inwhich an amino acid has been substituted into the antibody frameworkfrom the respective human VH or VL germline sequences.

Another aspect of the invention includes an isolated antibody or antigenbinding fragments thereof having the full heavy and light chainsequences of Fabs described in Table 1. More specifically, the isolatedantibody or antigen binding fragments thereof can have the heavy andlight chain sequences of NOV1090 and NOV1401.

A further aspect of the invention includes an isolated antibody orantigen binding fragments thereof having the heavy and light chainvariable domain sequences of Fabs described in Table 1. Morespecifically, the isolated antibody or antigen binding fragment thereofcan have the heavy and light chain variable domain sequence of NOV1090and NOV1401.

A further aspect of the invention includes an isolated antibody orantigen binding fragments thereof having the heavy chain variable domainCDR (i.e., HCDR1, HCDR2, and HCDR3) and light chain variable domain CDR(i.e., LCDR1, LCDR2, and LCDR3) sequences of antibodies described inTable 1, such as Kabat CDRs, IMGT CDRs, Chothia CDRs, or combined CDRs.More specifically, the isolated antibody or antigen binding fragmentthereof can have the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3sequences of NOV1090 and NOV1401, for example as presented in Table 1,such as Kabat CDRs, IMGT CDRs, Chothia CDRs, or combined CDRs.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that includes a heavy chain CDR1 selected from thegroup consisting of SEQ ID NOs: 3 and 23; a heavy chain CDR2 selectedfrom the group consisting of SEQ ID NOs: 4 and 24; and a heavy chainCDR3 selected from the group consisting of SEQ ID NOs: 5 and 25, whereinthe isolated antibody or antigen binding fragments thereof binds tohuman FXI and/or FXIa. In another aspect, such isolated antibody orantigen binding fragments thereof further includes a light chain CDR1selected from the group consisting of SEQ ID NOs: 13 and 33; a lightchain CDR2 selected from the group consisting of SEQ ID NOs: 14 and 34;and a light chain CDR3 selected from the group consisting of SEQ ID NOs:15 and 35.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that includes a light chain CDR1 selected from thegroup consisting of SEQ ID NOs: 13 and 33; a light chain CDR2 selectedfrom the group consisting of SEQ ID NOs: 14 and 34; and a light chainCDR3 selected from the group consisting of SEQ ID NOs: 15 and 35,wherein the isolated antibody or antigen binding fragments thereof bindsto human FXI and/or FXIa.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that binds FXI and/or FXIa having HCDR1, HCDR2, andHCDR3 and LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3comprises SEQ ID NOs: 3, 4, and 5, and LCDR1, LCDR2, LCDR3 comprises SEQID NOs: 13, 14, and 15; or HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs:23, 24, and 25, and LCDR1, LCDR2, LCDR3 comprises SEQ ID NOs: 33, 34,and 35.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that binds FXI and/or FXIa having HCDR1, HCDR2, andHCDR3 and LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3comprises SEQ ID NOs: 43, 44, and 45, respectively, and LCDR1, LCDR2,LCDR3 comprises SEQ ID NOs: 47, 37, and 15, respectively.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that binds FXI and/or FXIa having HCDR1, HCDR2, andHCDR3 and LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3comprises SEQ ID NOs: 46, 4, and 5, respectively, and LCDR1, LCDR2,LCDR3 comprises SEQ ID NOs: 33, 14, and 15, respectively.

The invention also relates to an antibody or antigen binding fragmenthaving HCDR1, HCDR2, and HCDR3 of the variable heavy chain of SEQ IDNOs: 9 and 29, and the LCDR1, LCDR2 and LCDR3 of the variable lightchain of SEQ ID NOs: 19 and 39, as defined by Chothia. In another aspectof the invention the antibody or antigen binding fragment may have theHCDR1, HCDR2, and HCDR3 of the heavy chain variable domain sequence ofSEQ ID NOs: 9 and 29, and the LCDR1, LCDR2 and LCDR3 of the light chainvariable domain sequence of SEQ ID NOs: 19 and 39, as defined by Kabat.

The invention also relates to an antibody or antigen binding fragmenthaving HCDR1, HCDR2, and HCDR3 of the variable heavy chain of SEQ IDNOs: 9 and 29, and the LCDR1, LCDR2 and LCDR3 of the variable lightchain of SEQ ID NOs: 19 and 39, as defined by IMGT. In another aspect ofthe invention the antibody or antigen binding fragment may have theHCDR1, HCDR2, and HCDR3 of the heavy chain variable domain sequence ofSEQ ID NOs: 9 and 29, and the LCDR1, LCDR2 and LCDR3 of the light chainvariable domain sequence of SEQ ID NOs: 19 and 39, as defined byCombined.

In one aspect of the invention the isolated antibody or antigen bindingfragments thereof includes a heavy chain variable domain sequenceselected from the group consisting of SEQ ID NOs: 9 and 29. The isolatedantibody or antigen binding fragment further can comprise a light chainvariable domain sequence wherein the heavy chain variable domain andlight chain variable domain combine to form an antigen binding site forFXIa. In particular the light chain variable domain sequence can beselected from SEQ ID NOs: 19 and 39 wherein said isolated antibody orantigen binding fragments thereof binds FXI and/or FXIa.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that includes a light chain variable domain sequenceselected from the group consisting of SEQ ID NOs: 19 and 39, whereinsaid isolated antibody or antigen binding fragments thereof binds tohuman FXI and/or FXIa. The isolated antibody or antigen binding fragmentmay further comprise a heavy chain variable domain sequence wherein thelight chain variable domain and heavy chain variable domain combine toform and antigen binding site for FXI and/or FXIa.

In particular, the isolated antibody or antigen binding fragmentsthereof that binds FXI and/or FXIa, may have heavy and light chainvariable domains comprising the sequences of SEQ ID NOs: 9 and 19; or 19and 39, respectively.

The invention further relates to an isolated antibody or antigen bindingfragments thereof, that includes a heavy chain variable domain having atleast 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to asequence selected from the group consisting of SEQ ID NOs: 9 and 29,wherein said antibody binds to FXI and/or FXIa. In one aspect, theisolated antibody or antigen binding fragments thereof also includes alight chain variable domain having at least 80%, 85%, 90%, 95%, 97%,98%, or 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 19 and 39. In a further aspect of theinvention, the isolated antibody or antigen binding fragment has anHCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as defined by Kabat and asdescribed in Table 1. In a specific embodiment, the isolated antibody orantigen binding fragment has an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 andLCDR3 as defined by Chothia, IMGT, or Combined and as described in Table1.

The invention also relates to an isolated antibody or antigen bindingfragments thereof, having a light chain variable domain having at least80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a sequenceselected from the group consisting of SEQ ID NOs: 19 and 39, whereinsaid antibody binds FXI and/or FXIa.

In another aspect of the invention, the isolated antibody, or antigenbinding fragments thereof, that bind to FXI and/or FXIa may have a heavychain comprising the sequence of SEQ ID NOs: 11 or 31. The isolatedantibody can also include a light chain that can combine with the heavychain to form an antigen binding site to human FXI and/or FXIa. Inparticular, the light chain may have a sequence comprising SEQ ID NOs:21 or 41. In particular, the isolated antibody or antigen bindingfragments thereof that binds FXI and/or FXIa, may have a heavy chain anda light chain comprising the sequences of SEQ ID NOs: 11 and 21; or 31and 41, respectively.

The invention still further relates to an isolated antibody or antigenbinding fragments thereof that includes a heavy chain having at least90% sequence identity to a sequence selected from the group consistingof SEQ ID NOs: 11 or 31, wherein said antibody binds to FXI and/or FXIa.In one aspect, the isolated antibody or antigen binding fragmentsthereof also includes a light chain having at least 80%, 85%, 90%, 95%,97%, 98%, or 99% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 21 or 41.

The invention still further relates to an isolated antibody or antigenbinding fragments thereof that includes a light chain having at least80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a sequenceselected from the group consisting of SEQ ID NOs: 21 or 41, wherein saidantibody binds FXI and/or FXIa.

The invention also relates to compositions comprising the isolatedantibody, or antigen binding fragments thereof, described herein, aswell as, antibody compositions in combination with a pharmaceuticallyacceptable carrier. Specifically, the invention further includespharmaceutical compositions comprising an antibody or antigen bindingfragments thereof of Table 1, such as, for example antibody NOV1090 andNOV1401. The invention also relates to pharmaceutical compositionscomprising a combination of two or more of the isolated antibodies orantigen binding fragments thereof of Table 1.

The invention also relates to an isolated nucleic acid sequence encodingthe variable heavy chain having a sequence selected from SEQ ID NOs: 9and 29. In particular the nucleic acid has a sequence at least 80%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 10 and 30. In a further aspect ofthe invention the sequence is SEQ ID NOs: 10 or 30.

The invention also relates to an isolated nucleic acid sequence encodingthe variable light chain having a sequence selected from SEQ ID NOs: 20and 40. In particular the nucleic acid has a sequence at least 80%, 85%,90%, 95%, 97%, 98%, or 99% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 20 and 40. In a further aspect ofthe invention the sequence is SEQ ID NOs: 20 and 40.

The invention also relates to an isolated nucleic acid comprising asequence encoding a polypeptide that includes a light chain variabledomain having at least 90% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 20 and 40.

The invention also relates to a vector that includes one or more of thenucleic acid molecules described herein.

The invention also relates to an isolated host cell that includes arecombinant DNA sequence encoding a heavy chain of the antibodydescribed above, and a second recombinant DNA sequence encoding a lightchain of the antibody described above, wherein said DNA sequences areoperably linked to a promoter and are capable of being expressed in thehost cell. It is contemplated that the antibody can be a humanmonoclonal antibody. It is also contemplated that the host cell is anon-human mammalian cell.

The invention also relates to a method of reducing FXI and/or FXIaexpression, and/or intrinsic and/or common coagulation pathwayactivation, wherein the method includes the step of contacting a cellwith an effective amount of a composition comprising the isolatedantibody or antigen binding fragments thereof described herein.

The invention also relates to a method of inhibiting the binding of FXIand/or FXIa to FIX, wherein the method includes the step of contacting acell with an effective amount of a composition comprising the isolatedantibody or antigen binding fragments thereof described herein.

It is contemplated that the cell is a human cell. It is furthercontemplated that the cell is in a subject. In one embodiment, it iscontemplated that the cell is a platelet. It is still furthercontemplated that the subject is human.

The invention also relates to a method of treating, improving, orpreventing a thromboembolic disease in a subject, wherein the methodincludes the step of administering to the subject an effective amount ofa composition comprising the antibody or antigen binding fragmentsthereof described herein. In one aspect, the thromboembolic disease is athrombotic disorder (e.g., thrombosis, thrombic stroke, atrialfibrillation, stroke prevention in atrial fibrillation (SPAF), deep veinthrombosis, venous thromboembolism, and pulmonary embolism). It iscontemplated that the subject is human.

Any of the foregoing isolated antibodies or antigen binding fragmentsthereof may be a monoclonal antibody or antigen binding fragmentsthereof.

Non-limiting embodiments of the disclosure are described in thefollowing aspects:

1. An isolated anti-FXI and/or anti-FXIa antibody or fragment thereofthat binds within the catalytic domain of FXI and/or FXIa.

2. An isolated antibody or fragment thereof that binds to one or moreepitopes of anti-FXI and/or FXIa, wherein the epitope comprises two ormore amino acid residues of Pro410, Arg413, Leu415, Cys416, His431,Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476,Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527, Arg548, His552,Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603, and Arg604.

3. The isolated antibody or fragment of aspect 2, wherein the epitopecomprises four or more amino acid residues of Pro410, Arg413, Leu415,Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474,Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527,Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603,and Arg604.

4. The isolated antibody or fragment of aspect 2, wherein the epitopecomprises six or more amino acid residues of Pro410, Arg413, Leu415,Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474,Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527,Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603,and Arg604.

5. The isolated antibody or fragment of aspect 2, wherein the epitopecomprises eight or more amino acid residues of Pro410, Arg413, Leu415,Cys416, His431, Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474,Ala475, Glu476, Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527,Arg548, His552, Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603,and Arg604.

6. The isolated antibody or fragment of aspect 2, wherein the epitopecomprises the residues of Pro410, Arg413, Leu415, Cys416, His431,Cys432, Tyr434, Gly435, Glu437, Tyr472, Lys473, Met474, Ala475, Glu476,Tyr521, Arg522, Lys523, Leu524, Arg525, Asp526, Lys527, Arg548, His552,Ser575, Ser594, Trp595, Gly596, Glu597, Arg602, Glu603, and Arg604.

7. The isolated antibody or fragment of aspect 2, wherein the epitopecomprises amino acid residues of Pro410, Arg413, Lys527 and one or moreamino acid residues of Leu415, Cys416, His431, Cys432, Tyr434, Gly435,Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523,Leu524, Arg525, Asp526, Arg548, His552, Ser575, Ser594, Trp595, Gly596,Glu597, Arg602, Glu603, and Arg604.

8. The isolated antibody or fragment of aspect 2, wherein the epitopecomprises amino acid residues of Pro410, Arg413, Lys527 and four or moreamino acid residues of Leu415, Cys416, His431, Cys432, Tyr434, Gly435,Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523,Leu524, Arg525, Asp526, Arg548, His552, Ser575, Ser594, Trp595, Gly596,Glu597, Arg602, Glu603, and Arg604.

9. The isolated antibody or fragment of aspect 2, wherein the epitopecomprises amino acid residues of Pro410, Arg413, Lys527 and six or moreamino acid residues of Leu415, Cys416, His431, Cys432, Tyr434, Gly435,Glu437, Tyr472, Lys473, Met474, Ala475, Glu476, Tyr521, Arg522, Lys523,Leu524, Arg525, Asp526, Arg548, His552, Ser575, Ser594, Trp595, Gly596,Glu597, Arg602, Glu603, and Arg604.

10. An isolated anti-FXI and/or anti-FXIa antibody or fragment thereofthat binds within the catalytic domain of FXI and/or FXIa, wherein saidantibody or fragment blocks FXI and/or FXIa binding to one or more ofFactor IX, Factor XIIa, and thrombin.

11. The isolated antibody or fragment of aspect 10, wherein saidantibody or fragment blocks FXI and/or FXIa binding to one or more ofFactor IX, Factor XIIa, or thrombin, and other components of thecoagulation pathway.

12. The isolated antibody or fragment of aspect 1, wherein said antibodyor fragment blocks one or more of FIX, FXI, and FXIa binding to plateletreceptors.

13. The isolated antibody or fragment of aspect 1, wherein said antibodyor fragment prevents activation of the intrinsic or common coagulationpathways.

14. An isolated antibody or fragment thereof that binds to a human FXIand/or FXIa protein with a K_(D) of less than or equal to 34 nM, asmeasured by BIACORE™ assay, or less than or equal to 4 pM, as measuredby solution equilibrium titration assay (SET).

15. The isolated antibody or fragment of aspect 1, wherein said antibodyor fragment comprises at least one complementarity determining regionhaving at least 90% identity to at least one of the CDRs recited inTable 1.

16. The isolated antibody or fragment of aspect 1, wherein said antibodyor fragment comprises a CDR1, CDR2, and CDR3 from Table 1.

17. An isolated variant of the antibody or fragment of aspect 1, whereinsaid antibody or fragment comprises a CDR1, CDR2, and CDR3 from Table 1,and wherein the variant has at least one to four amino acid changes inone of CDR1, CDR2, or CDR3.

18. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a heavy chain CDR3 selected from the groupconsisting of SEQ ID NO: 5 and 25.

20. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a VH selected from the group consisting of SEQ IDNO: 9 and 29 or an amino acid sequence with 90% identity thereof; and aVL selected from the group consisting of SEQ ID NO: 19 and 39 or anamino acid sequence with 90% identity thereof.

21. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a VH selected from the group consisting of SEQ IDNO: 9 and 29 or an amino acid sequence with 95% identity thereof; and aVL selected from the group consisting of SEQ ID NO: 19 and 39 or anamino acid sequence with 95% identity thereof.

22. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a VH selected from the group consisting of SEQ IDNO: 9 and 29 or an amino acid sequence with 97% identity thereof; and aVL selected from the group consisting of SEQ ID NO: 19 and 39 or anamino acid sequence with 97% identity thereof.

23. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a variable heavy chain sequence selected from thegroup consisting of SEQ ID NO: 9 and 29.

24. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a variable light chain sequence selected from thegroup consisting of SEQ ID NO: 19 and 39.

25. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a variable heavy chain selected from the groupconsisting of SEQ ID NO: 9 and 29; and variable light chain sequenceselected from the group consisting of SEQ ID NO: 19 and 39.

26. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment selected from the group consisting of an antibody orfragment comprising a variable heavy chain of SEQ ID NO: 9 and avariable light chain sequence of SEQ ID NO: 19 and an antibody orfragment comprising a variable heavy chain of SEQ ID NO: 29 and avariable light chain sequence of SEQ ID NO: 39.

27. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a heavy chain variable region CDR1 selected fromthe group consisting of SEQ ID NO: 46; CDR2 selected from the groupconsisting of SEQ ID NO: 4; CDR3 selected from the group consisting of5; a light chain variable region CDR1 selected from the group consistingof SEQ ID NO: 33; CDR2 selected from the group consisting of SEQ ID NO:14; and CDR3 selected from the group consisting of SEQ ID NO: 15.

28. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a heavy chain variable region CDR1 selected fromthe group consisting of SEQ ID NO: 3 and 23; CDR2 selected from thegroup consisting of SEQ ID NO: 4 and 24; CDR3 selected from the groupconsisting of 5 and 25; a light chain variable region CDR1 selected fromthe group consisting of SEQ ID NO: 13 and 33; CDR2 selected from thegroup consisting of SEQ ID NO: 14 and 34; and CDR3 selected from thegroup consisting of SEQ ID NO: 15 and 35.

29. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a heavy chain variable region CDR1 selected fromthe group consisting of SEQ ID NO: 6 and 26; CDR2 selected from thegroup consisting of SEQ ID NO: 7 and 27; CDR3 selected from the groupconsisting of 8 and 28; a light chain variable region CDR1 selected fromthe group consisting of SEQ ID NO: 16 and 36; CDR2 selected from thegroup consisting of SEQ ID NO: 17 and 37; and CDR3 selected from thegroup consisting of SEQ ID NO: 18 and 38.

30. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a heavy chain variable region CDR1 of SEQ ID NO:3; a heavy chain variable region CDR2 of SEQ ID NO: 4; a heavy chainvariable region CDR3 of SEQ ID NO: 5; a light chain variable region CDR1of SEQ ID NO: 13; a light chain variable region CDR2 of SEQ ID NO: 14;and a light chain variable region CDR3 of SEQ ID NO: 15.

31. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a heavy chain variable region CDR1 of SEQ ID NO:23; a heavy chain variable region CDR2 of SEQ ID NO: 24; a heavy chainvariable region CDR3 of SEQ ID NO: 25; a light chain variable regionCDR1 of SEQ ID NO: 33; a light chain variable region CDR2 of SEQ ID NO:34; and a light chain variable region CDR3 of SEQ ID NO: 35.

32. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a heavy chain variable region CDR1 of SEQ ID NO:6; a heavy chain variable region CDR2 of SEQ ID NO: 7; a heavy chainvariable region CDR3 of SEQ ID NO: 8; a light chain variable region CDR1of SEQ ID NO: 16; a light chain variable region CDR2 of SEQ ID NO: 17;and a light chain variable region CDR3 of SEQ ID NO: 18.

33. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment comprises a heavy chain variable region CDR1 of SEQ ID NO:26; a heavy chain variable region CDR2 of SEQ ID NO: 27; a heavy chainvariable region CDR3 of SEQ ID NO: 28; a light chain variable regionCDR1 of SEQ ID NO: 36; a light chain variable region CDR2 of SEQ ID NO:37; and a light chain variable region CDR3 of SEQ ID NO: 38.

34. A pharmaceutical composition comprising an antibody or fragmentthereof of one of the preceding aspects and a pharmaceuticallyacceptable carrier.

35. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment binds to the same epitope as an isolated antibody orfragment according to any previous aspect.

36. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment competes for binding to a human FXI and/or FXIa protein withan isolated antibody or fragment according to any previous aspect.

37. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment is selected from the group consisting of NOV1090 andNOV1401.

38. A method of treating a thromboembolic disorder comprisingadministering to a subject afflicted with a thromboembolic disorder aneffective amount of a pharmaceutical composition comprising an antibodyor fragment according to any previous aspect.

39. The method of aspect 38, wherein the subject is afflicated with oneor more of ischemic stroke associated with atrial fibrillation and deepvein thrombosis.

40. The method of aspect 38, wherein the subject is afflicated withischemic stroke associated with atrial fibrillation.

41. A method of treating a thromboembolic disorder comprisingadministering to a subject afflicted with a thromboembolic disorder aneffective amount of a pharmaceutical composition comprising an antibodyor fragment according to any previous aspect in combination with statintherapies.

42. A medicament comprising an antibody according to any previousaspect.

43. A nucleic acid coding for one or more of the antibodies according toany previous aspect.

44. A vector comprising the nucleic acid according to aspect 43.

45. A host cell comprising the vector of aspect 44.

46. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment leads upon binding to the active FXI (FXIa) catalytic domainto FXIa changing its conformation to an inactive conformation, in whichthe N-terminal 4 residues, loops 145, 188 and 220 are shifted and/ordisordered compared to the active conformation.

47. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment upon binding to FXI prevents the FXI catalytic domain fromassuming an active conformation, in which loops 145, 188 and 220 areordered as in the structure of the FXIa catalytic domain.

48. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment upon binding to FXI prevents the FXI catalytic domain fromassuming an active conformation, in which the N-terminal 4 residues,loops 145, 188 and 220 are ordered as in the structure of the FXIacatalytic domain.

49. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment upon binding to FXI prevents the FXI catalytic domain fromassuming an active conformation by inducing conformational changes inthe zymogen structure, further leading to an inhibited FXI conformationclosely related to that observed when binding to FXIa.

50. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment, upon binding to FXI and/or FXIa and forming an antibody:antigen complex with the catalytic domain of FXI and/or FXIa, causes ashift and/or disorientation of loops 145, 188 and 220 when compared withthe uncomplexed structure of the catalytic domain of active Factor XI(FXIa).

51. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment, upon binding to FXI and/or FXIa and forming an antibody:antigen complex with the catalytic domain of FXI and/or FXIa, causes ashift and/or disorientation of the N-terminal 4 residues, loops 145, 188and 220 when compared with the uncomplexed structure of the catalyticdomain of active Factor XI (FXIa).

52. The isolated antibody or fragment of aspect 1, wherein the antibodyor fragment binds to active FXI (FXIa) and causes the FXI (FXIa)catalytic domain to change its conformation to an inactive conformation,in which loops 145, 188 and 220 are shifted and/or disoriented comparedto the active conformation.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains.

The terms “FXI protein,” “FXI antigen,” and “FXI” are usedinterchangeably, and refers to the Factor XI protein in differentspecies. Factor XI is the mammalian plasma coagulation factor XI, aglycoprotein present in human plasma at a concentration of 25-30 nM as azymogen that when converted by limited proteolysis to an active serineprotease, participates in the intrinsic pathway of blood coagulation.

The terms “FXIa protein,” “FXIa antigen,” and “FXIa”, are usedinterchangeably, and refers to the activated FXI protein in differentspecies. The zymogen Factor XI is converted into its active form, thecoagulation factor XIa (FXIa), either via the contact phase of bloodcoagulation or through thrombin-mediated activation on the plateletsurface. During this activation of factor XI, an internal peptide bondis cleaved in each of the two chains, resulting in the activated factorXIa, a serine protease composed of two heavy and two light chains heldtogether by disulfide bonds. This serine protease FXIa converts thecoagulation Factor IX into IXa, which subsequently activates coagulationFactor X (Xa). Xa then can mediate coagulation Factor II/Thrombinactivation. For example, human FXI has the sequence as set out in Table1 (SEQ ID NO:1), and has been described in previous reports andliterature (Mandle R J Jr, et al. (1979) Blood; 54(4):850; NCBIReference Sequence: AAA51985).

In the context of this invention, the terms “FXI” and “FXIa” (and thelike) include mutants and variants of the natural FXI and FXIa protein,respectively, which have substantially the same amino acid sequence asthat of the native primary structure (amino acid sequence) described inthe above-mentioned reports.

The term “catalytic domain,” “serine protease catalytic domain,” andsimilar terms as used herein, means amino acids Ile370 to Va1607, ascounted from the Glu1 at the N-terminus of the mature protein that is incirculation. It can also be described as residues 388-625 at theC-terminus of FXI. As used herein, the term “active site” means thecatalytic triad comprised of the amino acids His413, Asp462 and Se557.(Bane and Gailani (2014) Drug Disc. 19(9)).

The term “about” in relation to a numerical value x means, for example,x±10%.

The term “antibody” as used herein means a whole antibody and anyantigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. A whole antibody is a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region is comprised of one domain, CL. The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The term “antigen binding portion” or “antigen binding fragment” of anantibody, as used herein, refers to one or more fragments of an intactantibody that retain the ability to specifically bind to a given antigen(e.g., Factor XIa (FXIa)). Antigen binding functions of an antibody canbe performed by fragments of an intact antibody. Examples of bindingfragments encompassed within the term antigen binding portion or antigenbinding fragment of an antibody include a Fab fragment, a monovalentfragment consisting of the VL, VH, CL and CH1 domains; a F(ab)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; an Fd fragment consisting of theVH and CH1 domains; an Fv fragment consisting of the VL and VH domainsof a single arm of an antibody; a single domain antibody (dAb) fragment(Ward et al., 1989 Nature 341:544-546), which consists of a VH domain ora VL domain; and an isolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by an artificial peptide linker that enables them to be made asa single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl.Acad. Sci. 85:5879-5883). Such single chain antibodies include one ormore antigen binding portions or fragments of an antibody. Theseantibody fragments are obtained using conventional techniques known tothose of skill in the art, and the fragments are screened for utility inthe same manner as are intact antibodies.

Antigen binding fragments can also be incorporated into single domainantibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies,tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005,Nature Biotechnology, 23, 9, 1126-1136). Antigen binding portions ofantibodies can be grafted into scaffolds based on polypeptides such asFibronectin type Ill (Fn3) (see U.S. Pat. No. 6,703,199, which describesfibronectin polypeptide monobodies).

Antigen binding fragments can be incorporated into single chainmolecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions (Zapata et al., 1995 Protein Eng.8(10):1057-1062; and U.S. Pat. No. 5,641,870).

As used herein, the term “affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity. As used herein,the term “high affinity” for an antibody or antigen binding fragmentsthereof (e.g., a Fab fragment) generally refers to an antibody, orantigen binding fragment, having a K_(D) of 10⁻⁹M or less (e.g., a K_(D)of 10⁻¹⁹M or less, a K_(D) of 10⁻¹¹M or less, a K_(D) of 10⁻¹² M orless, a K_(D) of 10⁻¹³ M or less, a K_(D) of 10⁻¹⁴ M or less, etc.).

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an alpha carbon that is boundto a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The term “binding specificity” as used herein refers to the ability ofan individual antibody combining site to react with only one antigenicdeterminant.

The phrase “specifically (or selectively) binds” to an antibody (e.g., aFXI and/or FXIa-binding antibody) refers to a binding reaction that isdeterminative of the presence of a cognate antigen (e.g., a human FXIand/or FXIa or cynomolgus FXI and/or FXIa) in a heterogeneous populationof proteins and other biologics. The phrases “an antibody recognizing anantigen” and “an antibody specific for an antigen” are usedinterchangeably herein with the term “an antibody which bindsspecifically to an antigen”.

The term “FXI and/or FXIa mediated” refers to the fact that FXI and/orFXIa mediates the intrinsic and/or common coagulation pathways bydirectly or indirectly activating Factor IX (also known as FIX), FactorX (FX), and/or thrombin, and/or by binding to platelet receptors.

The term “hemostasis” represents the principal mechanisms for arrestingthe flow of blood at sites of injury and restoring vascular patencyduring wound healing, respectively. During normal hemostasis andpathological thrombosis, three mechanisms become activatedsimultaneously: primary hemostasis meaning the interactions of activatedplatelets with the vessel wall, the formation of fibrin, and a processtermed as fibrinolysis.

The terms “coagulation and coagulation cascade,” “cascade model ofcoagulation,” and the like, refer to the protein based system whichserves to stabilize a clot that has formed to seal up a wound. Thecoagulation pathway is a proteolytic cascade. Each enzyme of the pathwayis present in the plasma as a Zymogen (in an inactive form), which onactivation undergoes proteolytic cleavage to release the active factorfrom the precursor molecule. The coagulation cascade functions as aseries of positive and negative feedback loops which control theactivation process. The ultimate goal of the pathway is to producethrombin, which can then convert soluble fibrinogen into fibrin thatforms a clot.

The process of generation of thrombin can be divided into three phases:the intrinsic and extrinsic pathways, which provide alternative routesfor the generation of an active clotting factor: FXa (ActivatedFactor-X), and the final common pathway, which results in thrombinformation (Hoffman M. M. and Monroe D. M. (2005) Curr Hematol Rep.4:391-396; Johne J, et al. (2006) Biol Chem. 387:173-178).

“Platelet aggregation” refers to the process whereby when a break in ablood vessel occurs, substances are exposed that normally are not indirect contact with the blood flow. These substances (primarily collagenand von Willebrand factor) allow the platelets to adhere to the brokensurface. Once a platelet adheres to the surface, it releases chemicalsthat attract additional platelets to the damaged area, referred to asplatelet aggregation. These two processes are the first responses tostop bleeding.

A “thromboembolic disorder,” or similar terms as used herein, refer toany number of conditions or diseases in which the intrinsic and/orcommon coagulation pathways are aberrantly activated or are notnaturally deactivated (e.g., without therapeutic means). Theseconditions include but are not limited to thrombic stroke, atrialfibrillation, stroke prevention in atrial fibrillation (SPAF), deep veinthrombosis, venous thromboembolism, and pulmonary embolism. These canalso include catheter-related conditions (e.g., Hickman catheter inoncology patients) in which catheters become thrombosed, andextracorporeal membrane oxygenation (ECMO), in which the tubing developsclots.

A “thromboembolic,” or similar terms as used herein, can also refer toany number of the following, which the anti-FXI and/or FXIa Abs orantigen binding fragments thereof of the invention can be used toprevent or treat:

-   -   thromboembolism in subjects with suspected or confirmed cardiac        arrhythmia such as paroxysmal, persistent or permanent atrial        fibrillation or atrial flutter;    -   stroke prevention in atrial fibrillation (SPAF), a subpopulation        of which is AF patients undergoing percutaneous coronary        interventions (PCI);    -   acute venous thromboembolic events (VTE) treatment and extended        secondary VTE prevention in patients at high risk for bleeding;    -   cerebral and cardiovascular events in secondary prevention after        transient ischemic attack (TIA) or non-disabling stroke and        prevention of thromboembolic events in heart failure with sinus        rhythm;    -   clot formation in left atrium and thromboembolism in subjects        undergoing cardioversion for cardiac arrhythmia;    -   thrombosis before, during and after ablation procedure for        cardiac arrhythmia;    -   venous thrombosis, this includes but not exclusively, treatment        and secondary prevention of deep or superficial veins thrombosis        in the lower members or upper member, thrombosis in the        abdominal and thoracic veins, sinus thrombosis and thrombosis of        jugular veins;    -   thrombosis on any artificial surface in the veins like catheter        or pacemaker wires;    -   pulmonary embolism in patients with or without venous        thrombosis;    -   Chronic Thromboembolic Pulmonary Hypertension (CTEPH);    -   arterial thrombosis on ruptured atherosclerotic plaque,        thrombosis on intra-arterial prosthesis or catheter and        thrombosis in apparently normal arteries, this includes but not        limited to acute coronary syndromes, ST elevation myocardial        infarction, non ST elevation myocardial infarction, unstable        angina, stent thrombosis, thrombosis of any artificial surface        in the arterial system and thrombosis of pulmonary arteries in        subjects with or without pulmonary hypertension;    -   thrombosis and thromboembolism in patients undergoing        percutaneous coronary interventions (PCI);    -   cardioembolic and cryptogenic strokes;    -   thrombosis in patients with invasive and non-invasive cancer        malignancies;    -   thrombosis over an indwelling catheter;    -   thrombosis and thromboembolism in severely ill patients;    -   cardiac thrombosis and thromboembolism, this includes but not        exclusively cardiac thrombosis after myocardial infarction,        cardiac thrombosis related to condition such as cardiac        aneurysm, myocardial fibrosis, cardiac enlargement and        insufficiency, myocarditis and artificial surface in the heart;    -   thromboembolism in patients with valvular heart disease with or        without atrial fibrillation;    -   thromboembolism over valvular mechanic or biologic prostheses;    -   thromboembolism in patients who had native or artificial cardiac        patches, arterial or venous conduit tubes after heart repair of        simple or complex cardiac malformations;    -   venous thrombosis and thromboembolism after knee replacement        surgery, hip replacement surgery, and orthopedic surgery,        thoracic or abdominal surgery;    -   arterial or venous thrombosis after neurosurgery including        intracranial and spinal cord interventions;    -   congenital or acquired thrombophilia including but not        exclusively factor V Leiden, prothrombin mutation, antithrombin        Ill, protein C and protein S deficiencies, factor XIII mutation,        familial dysfibrinogenemia, congenital deficiency of        plasminogen, increased levels of factor XI, sickle cell disease,        antiphospholipid syndrome, autoimmune disease, chronic bowel        disease, nephrotic syndrome, hemolytic uremia,        myeloproliferative disease, disseminated intra vascular        coagulation, paroxysmal nocturnal hemoglobinuria and heparin        induced thrombopenia;    -   thrombosis and thromboembolism in chronic kidney disease; and    -   thrombosis and thromboembolism in patients undergoing        hemodialysis and in patients undergoing extra-corporal membrane        oxygenation.

The term “chimeric antibody” is an antibody molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity. For example, a mouseantibody can be modified by replacing its constant region with theconstant region from a human immunoglobulin. Due to the replacement witha human constant region, the chimeric antibody can retain itsspecificity in recognizing the antigen while having reduced antigenicityin human as compared to the original mouse antibody.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. The following eight groups contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). In someembodiments, the term “conservative sequence modifications” are used torefer to amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. Two antibodies are said to “compete” if one antibody is shownto bind the same epitope as the second antibody in a competitive bindingassay, by any of the methods well known to those of skill in the art.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences. The human antibodies of theinvention may include amino acid residues not encoded by human sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or by somatic mutation in vivo).

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable regions in which both theframework and CDR regions are derived from human sequences. In oneembodiment, the human monoclonal antibodies are prepared using phagedisplay methods for screening libraries of human immunoglobulin genes.

A “humanized” antibody is an antibody that retains the reactivity of anon-human antibody while being less immunogenic in humans. This can beachieved, for instance, by retaining the non-human CDR regions andreplacing the remaining parts of the antibody with their humancounterparts (i.e., the constant region as well as the frameworkportions of the variable region). See, e.g., Morrison et al., Proc.Natl. Acad. Sci. USA, 81:6851-6855, 1984; Morrison and Oi, Adv.Immunol., 44:65-92, 1988; Verhoeyen et al., Science, 239:1534-1536,1988; Padlan, Molec. Immun., 28:489-498, 1991; and Padlan, Molec.Immun., 31:169-217, 1994. Other examples of human engineering technologyinclude, but are not limited to Xoma technology disclosed in U.S. Pat.No. 5,766,886.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of Pearson and Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Brent etal., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(Ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., (1977) Nuc. AcidsRes. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (VV) of11, an expectation (E) or 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17, 1988) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableon the world wide web at gcg.com), using either a Blossom 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “isolated antibody” refers to an antibody that is substantiallyfree of other antibodies having different antigenic specificities (e.g.,an isolated antibody that specifically binds FXI and/or FXIa issubstantially free of antibodies that specifically bind antigens otherthan FXI and/or FXIa). An isolated antibody that specifically binds FXIand/or FXIa may, however, have cross-reactivity to other antigens.Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The term “isotype” refers to the antibody class (e.g., IgM, IgE, IgGsuch as IgG1 or IgG4) that is provided by the heavy chain constantregion genes. Isotype also includes modified versions of one of theseclasses, where modifications have been made to alter the Fc function,for example, to enhance or reduce effector functions or binding to Fcreceptors.

The term “k_(assoc)” or “k_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “k_(dis)” or “k_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of k_(d) tok_(a) k_(d)/k_(a)) and is expressed as a molar concentration (M). K_(D)values for antibodies can be determined using methods well establishedin the art. Methods for determining the K_(D) of an antibody includemeasuring surface plasmon resonance using a biosensor system such as aBIACORE™ system, or measuring affinity in solution by solutionequilibrium titration (SET).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “nucleic acid” is used herein interchangeably with the term“polynucleotide” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, as detailed below,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem.260:2605-2608, 1985; and Rossolini et Mol. Cell. Probes 8:91-98, 1994).

The term “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, the termrefers to the functional relationship of a transcriptional regulatorysequence to a transcribed sequence. For example, a promoter or enhancersequence is operably linked to a coding sequence if it stimulates ormodulates the transcription of the coding sequence in an appropriatehost cell or other expression system. Generally, promotertranscriptional regulatory sequences that are operably linked to atranscribed sequence are physically contiguous to the transcribedsequence, i.e., they are cis-acting. However, some transcriptionalregulatory sequences, such as enhancers, need not be physicallycontiguous or located in close proximity to the coding sequences whosetranscription they enhance.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a Chinese Hamster Ovary cell (CHO)or a human cell. The optimized nucleotide sequence is engineered toretain completely or as much as possible the amino acid sequenceoriginally encoded by the starting nucleotide sequence, which is alsoknown as the “parental” sequence. The optimized sequences herein havebeen engineered to have codons that are preferred in mammalian cells.However, optimized expression of these sequences in other eukaryoticcells or prokaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

The term “recombinant host cell” (or simply “host cell”) refers to acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates (e.g.: mammals and non-mammals) such as,non-human primates (e.g.: cynomolgus monkey), sheep, rabbit, dog, cow,chickens, amphibians, and reptiles. Except when noted, the terms“patient” or “subject” are used herein interchangeably. As used herein,the terms “cyno” or “cynomolgus” refer to the cynomolgus monkey (Macacafascicularis). In specific aspects, a patient or subject is a human.

As used herein, the term “treating” or “treatment” of any disease ordisorder (e.g., a thromboembolic disorder) refers in one embodiment, toameliorating the disease or disorder (i.e., slowing or arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treating” or “treatment” refers to modulating thedisease or disorder, either physically, (e.g., stabilization of adiscernible symptom), physiologically, (e.g., stabilization of aphysical parameter), or both. In yet another embodiment, “treating” or“treatment” refers to preventing or delaying the onset or development orprogression of the disease or disorder.

“Prevention” as it relates to indications described herein, including,e.g., a thromboembolic disorder, means any action that prevents or slowsa worsening in e.g., a thromboembolic disease parameters, as describedbelow, in a patient at risk for said worsening.

The term “vector” is intended to refer to a polynucleotide moleculecapable of transporting another polynucleotide to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, such as anadeno-associated viral vector (AAV, or AAV2), wherein additional DNAsegments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the effect of NOV1401 on FeCl₃-induced thrombosis inFXI^(−/−) mice reconstituted with human FXI protein. NOV1401dose-dependently inhibited thrombosis. The antibody prolonged aPTT tothe same extent as in untreated FXI^(−/−) mice.

FIGS. 2A-B show the effect of multiple intravenous (i.v.) (A; N=2) orsubcutaneous (s.c.) (B; N=2) doses of 3 mg/kg, 10 mg/kg and 30 mg/kgNOV1401 on aPTT (diamonds) and relationship to total plasma NOV1401levels (squares) in cynomolgus monkeys. A single dose of 3 mg/kg led to˜2×aPTT that was maintained for 5-6 weeks. All doses tested prolongedaPTT to a similar extent, and the higher doses tested did not seem toincrease the magnitude of aPTT prolongation observed at the 3 mg/kgdose.

FIGS. 3A-B show the effect of multiple i.v. (A; N=2) or s.c. (B; N=2)doses of 3 mg/kg, 10 mg/kg and 30 mg/kg NOV1401 on plasma free FXI(squares) and relationship to aPTT (diamonds) in cynomolgus monkeys. Asingle dose of 3 mg/kg reduced free FXI by approximately 90% for 5-6weeks. All doses tested reduced free FXI to a similar extent, and thehigher doses tested did not seem to increase the magnitude of reductionof free FXI observed at the 3 mg/kg dose.

FIGS. 4A-B show the X-ray structure of the Fab of the NOV1401 antibodyof the invention bound to FXI. FIG. 4A shows the X-ray structure of theNOV1401 Fab-FXI CD complex. The FXI catalytic domain is shown as greysurface, the Fab as ribbon in light grey (light chain) and dark grey(heavy chain). FIG. 4B shows the X-ray structure of the NOV1401 Fab-FXICD complex in superposition with the FXI zymogen. The FXI catalyticdomain is shown as ribbon in grey. The variable domains of the Fab areshown as a ribbon in light gray (VL) and dark gray (VH). Superimposed isthe zymogen structure including the four apple domains as dark grayribbon at the structure's bottom (PDB 2F83). The activation cleavagesite (Ile370) is indicated.

FIGS. 5A-B shows structural changes of FXIa upon NOV1401 Fab binding.FIG. 5A shows a view of the FXIa active site prior to Fab binding. FXIais represented as a ribbon with a transparent surface. Sections of thestructure that change conformation upon Fab binding are labelled(loop145, loop188, and loop220). The S1 and S1′ subpockets areindicated. FIG. 5B shows the inactive conformation of FXI in theFab-complex (Fab not shown).

FIGS. 6A-C show compound response curves of an anti-FXI/FXIa antibody.FIG. 6A shows inhibition of Factor XIa activity by NOV1401.Representative compound response curve of antibody NOV1401 inhibitingthe enzymatic activity of full length human FXIa. The assay measures thecleavage of a fluorescently labelled peptide as is described in example3. Using the non-linear curve fit with a logistic fit model[y=A2+(A1−A2)/(1+(x/IC50)̂ p), where y is the %-inhibition at theinhibitor concentration, x. A1 is the lowest inhibition value, and A2the maximum inhibition value. The exponent, p, is the Hill coefficient]on this representative data set leads to an IC₅₀ value of 160 pM. FIG.6B shows an aPTT compound response curve. Representative compoundresponse curve of antibody NOV1401 prolonging coagulation time in theaPTT assay using pooled human plasma. The assay measures the time tocoagulation after initiating the intrinsic clotting cascade in presenceof different concentrations of NOV1401, as described in Example 4. Theblack line represents a fit using a logistics non-linear fit model. Thedotted line represents the baseline coagulation time of pooled humanplasma in absence of NOV1401. The baseline coagulation time is 32.3seconds, and is indicated with a grey dashed line in the graph. The greydotted line indicates the antibody concentration at which the clottingtime is doubled compared to baseline, i.e. the 2×aPTT value, which is 14nM. FIG. 6C shows a TGA response curve. A representative compoundresponse curve of antibody NOV1401 inhibiting thrombin generation in theTGA with pooled human plasma is shown. The assay measures the effects ofdifferent concentrations of NOV1401 on FXI-dependent thrombin generationthrough the so-called thrombing→FXIa feed-forward loop that can betriggered by very low tissue factor (TF) concentrations as described inExample 4. The black line represents a fit using a four-parameterdose-response curve model. The dotted line represents the residualthrombin concentration due to thrombin generation induced by smallamounts of TF. An IC₅₀ value of 24 nM and a residual thrombinconcentration of 159 nM (dotted line) were calculated for this compoundresponse curve.

FIGS. 7A-B show the effect of weekly NOV1401 doses of 10 mg/kg (N=3) and100 mg/kg (N=5) s.c. for 13 weeks (14 doses) or at 50 mg/kg (N=3) i.v.for 4 weeks (5 doses) on aPTT and FXI activity (FXI:C). FIG. 7A showsthe effect on aPTT, measured on study days 2, 23, and 79. aPTT increasedby 2.1- to 3-fold in all animals receiving NOV1401 and remained elevatedthroughout the dosing phase of the study. No dose-dependency wasobserved and no gender-related differences were noted. FIG. 7B shows theeffect on FXI:C, measured on study days 2, 23 and 79 and depicted aspercent of plasma FXI activity. FXI:C decreased in all animals receivingNOV1401 to levels of 5-12% and remained at these levels throughout thedosing phase of the study. No dose-dependency was observed and nogender-related differences were noted.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery of antibodymolecules that specifically bind to FXIa and inhibit its biologicalactivities. The invention relates to both full IgG format antibodies aswell as antigen binding fragments thereof, such as Fab fragments (e.g.,antibodies NOV1090 and NOV1401).

Accordingly, the present invention provides antibodies that specificallybind to FXI and/or FXIa (e.g., human, rabbit, and cynomolgus monkey FXIand/or FXIa), pharmaceutical compositions, production methods, andmethods of use of such antibodies and compositions.

Factor XI

FXI holds important roles in both intrinsic and extrinsic coagulationpathways and in bridging the initiation and amplification phases ofplasmatic hemostasis. Both Factor XIIa and thrombin can activate FXI,resulting in a sustained thrombin generation and fibrinolysisinhibition. FXI plays a minor role in normal hemostasis in a high tissuefactor environment “after vessel injury” whereas it appears to play akey role in thrombosis. Severe Factor XI deficiency is associated with alower incidence of ischemic stroke and venous thromboembolic events(Salomon et al 2008; Salomon, et al. (2011) Thromb Haemost.;105:269-73). Bleeding manifestations in subjects with severe factor XIdeficiency are infrequent, often mild, injury-induced and affectpreferably tissues with increased fibrinolytic activity such as the oralmucosa, nasal mucosa and urinary tract (Salomon et al 2011). Bleeding incritical organs is extremely rare or not existing.

Plasma coagulation is a sequential process by which coagulation factorsin the blood interact and are activated, ultimately resulting in fibringeneration and clot formation. In the classical cascade model ofcoagulation, the process of fibrin generation can be initiated by twodistinct pathways, i.e., the intrinsic and the extrinsic pathway,respectively (Mackman, 2008).

In the extrinsic pathway, vessel injury allows extravascular tissuefactor (TF) to interact with and activate factor VII (FVII), whichsequentially leads to the activation of factor X and prothrombin. Theactive thrombin ultimately converts soluble fibrinogen into fibrin. Theextrinsic pathway is central for hemostasis, interfering withcoagulation factors in this pathway results in a risk of bleeding.

In the intrinsic pathway, factor XII may in some cases be activated by aprocess referred to as contact activation. Generation of activatedfactor XIIa leads to the sequential activations of factor XI and factorIX. As factor IXa activates factor X, the extrinsic and intrinsicpathways converge at this stage (at the common pathway). Thrombinactivity is boosted by amplifying its own generation through afeed-forward loop in which thrombin activates factor XI independently offactor XII. This feed-forward loop contributes to sustained thrombusgrowth but is only minimally involved in hemostasis, as the strongactivation by extravascular tissue factor is sufficient to clotformation. The intrinsic pathway therefore is not substantially involvedin hemostasis (Gailani and Renne (2007) Arterioscler Thromb Vasc Biol.2007, 27(12):2507-13, Müller, Gailiani, and Renne 2011).

Preclinical studies using a variety of approaches to inhibit FXI or FXIaacross a variety of species have contributed to the validation of thistarget. FXI−/− mice are resistant to experimental venous (Wang, et al.(2006) J Thromb Haemost; 4:1982-8) and arterial (Wang, et al. (2005) JThromb Haemost; 3:695-702) thrombosis. Treatment of mice with anantibody (Ab, 14E11) that blocks the activation of FXI by FXIIa resultedin inhibition of experimental thrombosis (Cheng, et al. (2010) Blood,116:3981-9) and reduced cerebral infarct size in a mouse model ofischemic stroke (Leung, et al. (2012) Transl Stroke Res 2012; 3:381-9).In baboons administered an anti-FXI Ab that blocks binding andactivation of FIX by FXIa, reduced growth of platelet-rich thrombi wasobserved on collagen-coated vascular grafts (Tucker, et al. (2009) Blood2009; 113:936-44), and similar results were found with 14E11 in thismodel (Cheng 2010). Excessive bleeding was not noted in any of thesestudies.

Blocking FXI synthesis with antisense oligonucleotides in mice (Zhang,et al. (2010) Blood 2010; 116:4684-92), cynomolgus monkeys (Younis, etal. (2012) Blood 2012; 119:2401-8), and baboons (Crosby, et al. (2013)Arterioscler Thromb Vasc Biol 2013; 33:1670-8) resulted inantithrombotic and anticoagulant effects without excessive bleeding.Moreover, similar effects have been produced by blocking FXIa with lowmolecular weight inhibitors in venous and arterial models of thrombosisin rats (Schumacher, et al. (2007) Eur J Pharmacol 2007; 570:167-74) andrabbits (Wong, et al. (2011) J Thromb Thrombolysis 2011; 32:129-37).

Patients with severe FXI deficiency rarely bleed spontaneously and theyshow only mild trauma-induced bleeding, except in tissues with highfibrinolytic activity. The rarity of severe FXI deficiency necessitatesthe use of population studies for revealing the thrombotic profile ofthese patients relative to the general population. Notably, such studiesreport the incidence of ischemic stroke (Salomon 2008) and deep veinthrombosis (DVT) (Salomon, et al. (2011) Blood 2008; 111: 4113-17) to bereduced in these patients. Thus, the number of ischemic strokes (N=1)observed in 115 patients with severe FXI deficiency was less (p<0.003)than the expected incidence (N=8.6) in the general population of Israel,while the incidence of DVT (N=0) was lower (p<0.019) in patients withsevere FXI deficiency than expected in the control population (N=4.7).Conversely, individuals with FXI levels above the 90th percentile had atwo-fold risk of developing DVT (Meijers, et al. (2000) N Engl J Med.2000; 342:696-701).

Recently, patients undergoing total knee replacement, a procedure thatpredisposes to DVT, were treated with FXI antisense therapy or standardof care (enoxaparin). The antisense group (300 mg) showed a 7-folddecreased incidence in venous thrombosis and fewer (not significant)bleeding events compared to standard of care (Buller et al, (2014) NEngl J Med. 372(3):232-40. doi: 10.1056/NEJMoa1405760. Epub 2014 Dec.7).

Taken together, the above studies strongly support FXI as a valid targetfor antithrombotic therapy.

FXIa Antibodies & Antigen Binding Fragments

The present invention provides antibodies that specifically bind to FXIand/or FXIa. In some embodiments, the present invention providesantibodies that specifically bind to human, rabbit, and cynomolgusmonkey FXI and/or FXIa. Antibodies of the invention include, but are notlimited to, the human monoclonal antibodies and Fabs, isolated asdescribed in the Examples.

The present invention provides antibodies that specifically bind a FXIand/or FXIa protein (e.g., human, rabbit, and cynomolgus monkey FXIand/or FXIa), wherein the antibodies comprise a VH domain having anamino acid sequence of SEQ ID NOs: 9 and 29. The present invention alsoprovides antibodies that specifically bind to a FXI and/or FXIa protein,wherein the antibodies comprise a VH CDR having an amino acid sequenceof any one of the VH CDRs listed in Table 1, infra. In particular, theinvention provides antibodies that specifically bind to an FXI and/orFXIa protein (e.g., human, rabbit, and cynomolgus monkey FXI and/orFXIa), wherein the antibodies comprise (or alternatively, consist of)one, two, three, or more VH CDRs having an amino acid sequence of any ofthe VH CDRs listed in Table 1, infra.

The present invention provides antibodies that specifically bind to aFXIa protein, said antibodies comprising a VL domain having an aminoacid sequence of SEQ ID NOs: 19 or 39. The present invention alsoprovides antibodies that specifically bind to an FXI and/or FXIa protein(e.g., human, rabbit, and cynomolgus monkey FXI and/or FXIa), saidantibodies comprising a VL CDR having an amino acid sequence of any oneof the VL CDRs listed in Table 1, infra. In particular, the inventionprovides antibodies that specifically bind to an FXIa protein (e.g.,human, rabbit, and cynomolgus monkey FXI and/or FXIa), said antibodiescomprising (or alternatively, consisting of) one, two, three or more VLCDRs having an amino acid sequence of any of the VL CDRs listed in Table1, infra.

Other antibodies of the invention include amino acids that have beenmutated, yet have at least 60, 70, 80, 85, 90 or 95 percent identity inthe CDR regions with the CDR regions depicted in the sequences describedin Table 1. In some embodiments, it includes mutant amino acid sequenceswherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated inthe CDR regions when compared with the CDR regions depicted in thesequence described in Table 1.

The present invention also provides nucleic acid sequences that encodeVH, VL, the full length heavy chain, and the full length light chain ofthe antibodies that specifically bind to a FXI and/or FXIa protein(e.g., human, rabbit, and cynomolgus monkey FXIa). Such nucleic acidsequences can be optimized for expression in mammalian cells (forexample, Table 1 shows the optimized nucleic acid sequences for theheavy chain and light chain of antibodies of the invention).

TABLE 1 Examples of FXIa Antibodies, Fabs and FXIa Proteins. Sequence Identifier (SEQ ID Sequence Description NO:)Amino acid or polynucleotide sequence Human FXIa full-  1MIFLYQVVHF ILFTSVSGEC VTQLLKDTCF EGGDITTVFT length proteinPSAKYCQVVC TYHPRCLLFT FTAESPSEDP TRWFTCVLKD sequence (NCBISVTETLPRVN RTAAISGYSF KQCSHQISAC NKDIYVDLDM Reference Sequence:KGINYNSSVA KSAQECQERC TDDVHCHFFT YATRQFPSLE AAA51985)HRNICLLKHT QTGTPTRITK LDKVVSGFSL KSCALSNLACIRDIFPNTVF ADSNIDSVMA PDAFVSGRIC THHPGCLFFTFFSQEWPKES QRNLCLLKTS ESGLPSTRIK KSKALSGFSLQSCRHSIPVF CHSSFYHDTD FLGEELDIVA AKSHEACQKLCTNAVRCQFF TYTPAQASCN EGKGKCYLKL SSNGSPTKILHGRGGISGYT LRLCKMDNEC TTKIKPRIVG GTASVRGEWPWQVTLHTTSP TQRHLCGGSI IGNQWILTAA HCFYGVESPKILRVYSGILN QSEIKEDTSF FGVQEIIIHD QYKMAESGYDIALLKLETTV NYTDSQRPIC LPSKGDRNVI YTDCWVTGWGYRKLRDKIQN TLQKAKIPLV TNEECQKRYR GHKITHKMICAGYREGGKDA CKGDSGGPLS CKHNEVWHLV GITSWGEGCA QRERPGVYTN VVEYVDWILE KTQAVHuman FXIa full-  2 AGGCACACAG GCAAAATCAA GTTCTACATC TGTCCCTGTGlength nucleotide TATGTCACTT GTTTGAATAC GAAATAAAAT TAAAAAAATAsequence (NCBI AATTCAGTGT ATTGAGAAAG CAAGCAATTC TCTCAAGGTAReference Sequence: TATTTCTGAC ATACTAAGAT TTTAACGACT TTCACAAATANM_000128.3) TGCTGTACTG AGAGAGAATG TTACATAACA TTGAGAACTAGTACAAGTAA ATATTAAAGT GAAGTGACCA TTTCCTACACAAGCTCATTC AGAGGAGGAT GAAGACCATT TTGGAGGAAGAAAAGCACCC TTATTAAGAA TTGCAGCAAG TAAGCCAACAAGGTCTTTTC AGGATGATTT TCTTATATCA AGTGGTACATTTCATTTTAT TTACTTCAGT TTCTGGTGAA TGTGTGACTCAGTTGTTGAA GGACACCTGC TTTGAAGGAG GGGACATTACTACGGTCTTC ACACCAAGCG CCAAGTACTG CCAGGTAGTCTGCACTTACC ACCCAAGATG TTTACTCTTC ACTTTCACGGCGGAATCACC ATCTGAGGAT CCCACCCGAT GGTTTACTTGTGTCCTGAAA GACAGTGTTA CAGAAACACT GCCAAGAGTGAATAGGACAG CAGCGATTTC TGGGTATTCT TTCAAGCAATGCTCACACCA AATAAGCGCT TGCAACAAAG ACATTTATGTGGACCTAGAC ATGAAGGGCA TAAACTATAA CAGCTCAGTTGCCAAGAGTG CTCAAGAATG CCAAGAAAGA TGCACGGATGACGTCCACTG CCACTTTTTC ACGTACGCCA CAAGGCAGTTTCCCAGCCTG GAGCATCGTA ACATTTGTCT ACTGAAGCACACCCAAACAG GGACACCAAC CAGAATAACG AAGCTCGATAAAGTGGTGTC TGGATTTTCA CTGAAATCCT GTGCACTTTCTAATCTGGCT TGTATTAGGG ACATTTTCCC TAATACGGTGTTTGCAGACA GCAACATCGA CAGTGTCATG GCTCCCGATGCTTTTGTCTG TGGCCGAATC TGCACTCATC ATCCCGGTTGCTTGTTTTTT ACCTTCTTTT CCCAGGAATG GCCCAAAGAATCTCAAAGAA ATCTTTGTCT CCTTAAAACA TCTGAGAGTGGATTGCCCAG TACACGCATT AAAAAGAGCA AAGCTCTTTCTGGTTTCAGT CTACAAAGCT GCAGGCACAG CATCCCAGTGTTCTGCCATT CTTCATTTTA CCATGACACT GATTTCTTGGGAGAAGAACT GGATATTGTT GCTGCAAAAA GTCACGAGGCCTGCCAGAAA CTGTGCACCA ATGCCGTCCG CTGCCAGTTTTTTACCTATA CCCCAGCCCA AGCATCCTGC AACGAAGGGAAGGGCAAGTG TTACTTAAAG CTTTCTTCAA ACGGATCTCCAACTAAAATA CTTCACGGGA GAGGAGGCAT CTCTGGATACACATTAAGGT TGTGTAAAAT GGATAATGAG TGTACCACCAAAATCAAGCC CAGGATCGTT GGAGGAACTG CGTCTGTTCGTGGTGAGTGG CCGTGGCAGG TGACCCTGCA CACAACCTCACCCACTCAGA GACACCTGTG TGGAGGCTCC ATCATTGGAAACCAGTGGAT ATTAACAGCC GCTCACTGTT TCTATGGGGTAGAGTCACCT AAGATTTTGC GTGTCTACAG TGGCATTTTAAATCAATCTG AAATAAAAGA GGACACATCT TTCTTTGGGGTTCAAGAAAT AATAATCCAT GATCAGTATA AAATGGCAGAAAGCGGGTAT GATATTGCCT TGTTGAAACT GGAAACCACAGTGAATTACA CAGATTCTCA ACGACCCATA TGCCTGCCTTCCAAAGGAGA TAGAAATGTA ATATACACTG ATTGCTGGGTGACTGGATGG GGGTACAGAA AACTAAGAGA CAAAATACAAAATACTCTCC AGAAAGCCAA GATACCCTTA GTGACCAACGAAGAGTGCCA GAAGAGATAC AGAGGACATA AAATAACCCATAAGATGATC TGTGCCGGCT ACAGGGAAGG AGGGAAGGACGCTTGCAAGG GAGATTCGGG AGGCCCTCTG TCCTGCAAACACAATGAGGT CTGGCATCTG GTAGGCATCA CGAGCTGGGGCGAAGGCTGT GCTCAAAGGG AGCGGCCAGG TGTTTACACCAACGTGGTCG AGTACGTGGA CTGGATTCTG GAGAAAACTCAAGCAGTGTG AATGGGTTCC CAGGGGCCAT TGGAGTCCCTGAAGGACCCA GGATTTGCTG GGAGAGGGTG TTGAGTTCACTGTGCCAGCA TGCTTCCTCC ACAGTAACAC GCTGAAGGGGCTTGGTGTTT GTAAGAAAAT GCTAGAAGAA AACAAACTGTCACAAGTTGT TATGTCCAAA ACTCCCGTTC TATGATCGTTGTAGTTTGTT TGAGCATTCA GTCTCTTTGT TTTTGATCACGCTTCTATGG AGTCCAAGAA TTACCATAAG GCAATATTTCTGAAGATTAC TATATAGGCA GATATAGCAG AAAATAACCAAGTAGTGGCA GTGGGGATCA GGCAGAAGAA CTGGTAAAAGAAGCCACCAT AAATAGATTT GTTCGATGAA AGATGAAAACTGGAAGAAAG GAGAACAAAG ACAGTCTTCA CCATTTTGCAGGAATCTACA CTCTGCCTAT GTGAACACAT TTCTTTTGTAAAGAAAGAAA TTGATTGCAT TTAATGGCAG ATTTTCAGAATAGTCAGGAA TTCTTGTCAT TTCCATTTTA AAATATATATTAAAAAAAAT CAGTTCGAGT AGACACGAGC TAAGAGTGAATGTGAAGATA ACAGAATTTC TGTGTGGAAG AGGATTACAAGCAGCAATTT ACCTGGAAGT GATACCTTAG GGGCAATCTTGAAGATACAC TTTCCTGAAA AATGATTTGT GATGGATTGTATATTTATTT AAAATATCTT GGGAGGGGAG GCTGATGGAGATAGGGAGCA TGCTCAAACC TCCCTAAGAC AAGCTGCTGCTGTGACTATG GGCTCCCAAA GAGCTAGATC GTATATTTATTTGACAAAAA TCACCATAGA CTGCATCCAT ACTACAGAGAAAAAACAATT AGGGCGCAAA TGGATAGTTA CAGTAAAGTCTTCAGCAAGC AGCTGCCTGT ATTCTAAGCA CTGGGATTTTCTGTTTCGTG CAAATATTTA TCTCATTATT GTTGTGATCTAGTTCAATAA CCTAGAATTT GAATTGTCAC CACATAGCTTTCAATCTGTG CCAACAACTA TACAATTCAT CAAGTGTG NOV1090 HCDR1 (Kabat)  3 TAAMSHCDR2 (Kabat)  4 GISGSGSSTYYADSVKG HCDR3 (Kabat)  5 ELSYLYSGYYFDYHCDR1 (Chothia)  6 GFTFSTA HCDR2 (Chothia)  7 SGSGSS HCDR3 (Chothia)  8ELSYLYSGYYFDY HCDR1 (IMGT) 43 GFTFSTAA HCDR2 (IMGT) 44 ISGSGSSTHCDR3 (IMGT) 45 ARELSYLYSGYYFDY HCDR1 (Combined) 46 GFTFSTAAMSHCDR2 (Combined)  4 GISGSGSSTYYADSVKG HCDR3 (Combined)  5 ELSYLYSGYYFDYVH  9 QVQLLESGGGLVQPGGSLRLSCAASGFTFSTAAMSWVRQAPGKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELSYLYSGYYFDYWGQGTLVTVSS DNA encoding VH 10CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCCGGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCACCTTTTCTACTGCTGCTATGTCTTGGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGTGGGTTTCCGGTATCTCTGGTTCTGGTTCTTCTACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACCATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGAACTGTCTTACCTGTACTCTGGTTACTACTTCGATTACTGGGGCCAAGG CACCCTGGTGACTGTTAGCTCAHeavy Chain 11 QVQLLESGGGLVQPGGSLRLSCAASGFTFSTAAMSWVRQAPGKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELSYLYSGYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGKDNA encoding Heavy 12 CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCCGG ChainGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCACCTTTTCTACTGCTGCTATGTCTTGGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGTGGGTTTCCGGTATCTCTGGTTCTGGTTCTTCTACCTACTATGCGGATAGCGTGAAAGGCCGCTTTACCATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGAACTGTCTTACCTGTACTCTGGTTACTACTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCAGCGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA GCCTCTCCCTGTCTCCGGGTAAALCDR1 (Kabat) 13 SGSSSNIGSNDVS LCDR2 (Kabat) 14 KNYNRPS LCDR3 (Kabat) 15SAWDQRQFDVV LCDR1 (Chothia) 16 SSSNIGSND LCDR2 (Chothia) 17 KNYLCDR3 (Chothia) 18 WDQRQFDV LCDR1 (IMGT) 47 SSNIGSND LCDR2 (IMGT) 37 KNYLCDR3 (IMGT) 15 SAWDQRQFDVV LCDR1 (Combined) 33 SGSSSNIGSNDVSLCDR2 (Combined) 14 KNYNRPS LCDR3 (Combined) 15 SAWDQRQFDVV VL 19DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYKNYNRPSGVPDRFSGSKSGTSASLAITGLQAEDEAD YYCSAWDQRQFDVVFGGGTKLTVLDNA encoding VL 20 GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACCGGGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCAACATTGGTTCTAACGACGTGTCTTGGTACCAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTACAAAAACTACAACCGCCCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTACTGCTCTGCTTGGGACCAGCGTCAGTTCGACGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTA Light Chain 21DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYKNYNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCSAWDQRQFDVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC S DNA encoding Light 22GATATCGTGCTGACCCAGCCGCCGAGCGTGAGCGGTGCACCGG ChainGCCAGCGCGTGACCATTAGCTGTAGCGGCAGCAGCAGCAACATTGGTTCTAACGACGTGTCTTGGTACCAGCAGCTGCCGGGCACGGCGCCGAAACTGCTGATCTACAAAAACTACAACCGCCCGAGCGGCGTGCCGGATCGCTTTAGCGGATCCAAAAGCGGCACCAGCGCCAGCCTGGCGATTACCGGCCTGCAAGCAGAAGACGAAGCGGATTATTACTGCTCTGCTTGGGACCAGCGTCAGTTCGACGTTGTGTTTGGCGGCGGCACGAAGTTAACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGT TCA NOV1401 HCDR1 (Kabat) 23TAAMS HCDR2 (Kabat) 24 GISGSGSSTYYADSVKG HCDR3 (Kabat) 25 ELSYLYSGYYFDYHCDR1 (Chothia) 26 GFTFSTA HCDR2 (Chothia) 27 SGSGSS HCDR3 (Chothia) 28ELSYLYSGYYFDY HCDR1 (IMGT) 43 GFTFSTAA HCDR2 (IMGT) 44 ISGSGSSTHCDR3 (IMGT) 45 ARELSYLYSGYYFDY HCDR1 (Combined) 46 GFTFSTAAMSHCDR2 (Combined)  4 GISGSGSSTYYADSVKG HCDR3 (Combined)  5 ELSYLYSGYYFDYVH 29 QVQLLESGGGLVQPGGSLRLSCAASGFTFSTAAMSWVRQAPGKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELSYLYSGYYFDYWGQGTLVTVSS DNA encoding VH 30CAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTTAGCACCGCCGCTATGAGCTGGGTTCGACAGGCCCCAGGGAAAGGCCTCGAGTGGGTCTCAGGGATTAGCGGTAGCGGCTCTAGCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCACTATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTCTACTACTGCGCTAGAGAGCTGAGCTACCTGTATAGCGGCTACTACTTCGACTACTGGGGTCAAGG CACCCTGGTCACCGTGTCTAGCHeavy Chain 31 QVQLLESGGGLVQPGGSLRLSCAASGFTFSTAAMSWVRQAPGKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELSYLYSGYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGKDNA encoding Heavy 32 CAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCCTG ChainGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTTAGCACCGCCGCTATGAGCTGGGTTCGACAGGCCCCAGGGAAAGGCCTCGAGTGGGTCTCAGGGATTAGCGGTAGCGGCTCTAGCACCTACTACGCCGATAGCGTGAAGGGCCGGTTCACTATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTCTACTACTGCGCTAGAGAGCTGAGCTACCTGTATAGCGGCTACTACTTCGACTACTGGGGTCAAGGCACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAACTGCTGGGCGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGGCCGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGT CCCTGTCCCTGTCTCCCGGCAAGLCDR1 (Kabat) 33 SGSSSNIGSNDVS LCDR2 (Kabat) 34 KNYNRPS LCDR3 (Kabat) 35SAWDQRQFDVV LCDR1 (Chothia) 36 SSSNIGSND LCDR2 (Chothia) 37 KNYLCDR3 (Chothia) 38 WDQRQFDV LCDR1 (IMGT) 47 SSNIGSND LCDR2 (IMGT) 37 KNYLCDR3 (IMGT) 15 SAWDQRQFDVV LCDR1 (Combined) 33 SGSSSNIGSNDVSLCDR2 (Combined) 14 KNYNRPS LCDR3 (Combined) 15 SAWDQRQFDVV VL 39QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYKNYNRPSGVPDRFSGSKSGTSASLAISGLQSEDEAD YYCSAWDQRQFDVVFGGGTKLTVLDNA encoding VL 40 CAGTCAGTCCTGACTCAGCCCCCTAGCGCTAGTGGCACCCCTGGTCAAAGAGTGACTATTAGCTGTAGCGGCTCTAGCTCTAATATCGGCTCTAACGACGTCAGCTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTATAAGAACTATAATAGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGATCTAAATCAGGGACTTCTGCTAGTCTGGCTATTAGCGGCCTGCAGTCAGAGGACGAGGCCGACTACTACTGTAGCGCCTGGGATCAGCGTCAGTTCGACGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTG Light Chain 41QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYKNYNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCSAWDQRQFDVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC S DNA encoding Light 42CAGTCAGTCCTGACTCAGCCCCCTAGCGCTAGTGGCACCCCTG ChainGTCAAAGAGTGACTATTAGCTGTAGCGGCTCTAGCTCTAATATCGGCTCTAACGACGTCAGCTGGTATCAGCAGCTGCCCGGCACCGCCCCTAAGCTGCTGATCTATAAGAACTATAATAGGCCTAGCGGCGTGCCCGATAGGTTTAGCGGATCTAAATCAGGGACTTCTGCTAGTCTGGCTATTAGCGGCCTGCAGTCAGAGGACGAGGCCGACTACTACTGTAGCGCCTGGGATCAGCGTCAGTTCGACGTGGTGTTCGGCGGAGGCACTAAGCTGACCGTGCTGGGTCAACCTAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAAAAGACCGTGGCCCCAACCGAGTGC AGC

Other antibodies of the invention include those where the amino acids ornucleic acids encoding the amino acids have been mutated, yet have atleast 60, 65, 70, 75, 80, 85, 90, or 95 percent identity to thesequences described in Table 1. Some embodiments include mutant aminoacid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids havebeen mutated in the variable regions when compared with the variableregions depicted in the sequence described in Table 1, while retainingsubstantially the same antigen binding activity.

Since each of these antibodies can bind to FXI and/or FXIa, the VH, VL,full length light chain, and full length heavy chain sequences (aminoacid sequences and the nucleotide sequences encoding the amino acidsequences) can be “mixed and matched” to create other FXI and/orFXIa-binding antibodies of the invention. Such “mixed and matched” FXIand/or FXIa-binding antibodies can be tested using the binding assaysknown in the art (e.g., ELISAs, and other assays described in theExample section). When these chains are mixed and matched, a VH sequencefrom a particular VH/VL pairing should be replaced with a structurallysimilar VH sequence. Likewise a full length heavy chain sequence from aparticular full length heavy chain/full length light chain pairingshould be replaced with a structurally similar full length heavy chainsequence. Likewise, a VL sequence from a particular VH/VL pairing shouldbe replaced with a structurally similar VL sequence. Likewise a fulllength light chain sequence from a particular full length heavychain/full length light chain pairing should be replaced with astructurally similar full length light chain sequence.

Accordingly, in one aspect, the invention provides an isolated antibodyor antigen binding region thereof having: a heavy chain variable domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 9 and 29, and a light chain variable domain comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 19and 39, wherein the antibody specifically binds to FXI and/or FXIa(e.g., human, rabbit, and cynomolgus monkey FXIa).

More specifically, in certain aspects, the invention provides anisolated antibody or antigen binding region thereof having a heavy chainvariable domain and a light chain variable domain comprising amino acidsequences selected from SEQ ID NOs: 9 and 29; or 19 and 39,respectively.

In a specific embodiment, an antibody or antigen binding fragmentthereof provided herein which specifically binds to human FXI and/orFXIa, comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 9, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 19.

In a specific embodiment, an antibody or antigen binding fragmentthereof provided herein which specifically binds to human FXI and/orFXIa, comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 29, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 39.

In another aspect, the invention provides (i) an isolated antibodyhaving: a full length heavy chain comprising an amino acid sequence thathas been optimized for expression in a mammalian cell selected from thegroup consisting of SEQ ID NOs: 11 or 31, and a full length light chaincomprising an amino acid sequence that has been optimized for expressionin a mammalian cell selected from the group consisting of SEQ ID NOs: 21or 41; or (ii) a functional protein comprising an antigen bindingportion thereof. More specifically, in certain aspects, the inventionprovides an isolated antibody or antigen binding region thereof having aheavy chain and a light chain comprising amino acid sequences selectedfrom SEQ ID NOs: 11 and 31; or 19 and 39, respectively.

In a specific embodiment, an antibody or antigen binding fragmentthereof provided herein which specifically binds to human FXI and/orFXIa, comprises a heavy chain comprising the amino acid sequence of SEQID NO: 11, and a light chain comprising the amino acid sequence of SEQID NO: 21.

In a specific embodiment, an antibody or antigen binding fragmentthereof provided herein which specifically binds to human FXI and/orFXIa, comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 31, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 41.

The terms “complementarity determining region,” and “CDR,” as usedherein refer to the sequences of amino acids within antibody variableregions which confer antigen specificity and binding affinity. Ingeneral, there are three CDRs in each heavy chain variable region(HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region(LCDR1, LCDR2, LCDR3).

The precise amino acid sequence boundaries of a given CDR can be readilydetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme),Lefranc et al., (2003) Dev. Comp. Immunol., 27, 55-77 (“IMGT” numberingscheme), or the “Combined” system.

For example, under Kabat, the CDR amino acid residues of antibodyNOV1090 in the heavy chain variable domain (VH) are numbered 31-35(HCDR1), 50-66 (HCDR2), and 99-111 (HCDR3); and the CDR amino acidresidues in the light chain variable domain (VL) are numbered 22-35(LCDR1), 51-57 (LCDR2), and 90-100 (LCDR3). Under Chothia the CDR aminoacids in the VH are numbered 26-32 (HCDR1), 52-57 (HCDR2), and 99-111(HCDR3); and the amino acid residues in VL are numbered 25-33 (LCDR1),51-53 (LCDR2), and 92-99 (LCDR3). By combining the CDR definitions ofboth Kabat and Chothia, the CDRs consist of amino acid residues 26-35(HCDR1), 50-66 (HCDR2), and 99-111 (HCDR3) in human VH and amino acidresidues 22-35 (LCDR1), 51-57 (LCDR2), and 90-100 (LCDR3) in human VL.By combining the CDR definitions of both Kabat and Chothia, the“Combined” CDRs consist of amino acid residues 26-35 (HCDR1), 50-66(HCDR2), and 99-108 (HCDR3) in human VH and amino acid residues 24-38(LCDR1), 54-60 (LCDR2), and 93-101 (LCDR3) in human VL. As anotherexample, under IMGT, the CDR amino acid residues in the heavy chainvariable domain (VH) are numbered 26-33 (HCDR1), 51-58 (HCDR2), and97-108 (HCDR3); and the CDR amino acid residues in the light chainvariable domain (VL) are numbered 27-36 (LCDR1), 54-56 (LCDR2), and93-101 (LCDR3). Table 1 provides exemplary Kabat, Chothia, Combined, andIMGT HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 for anti-FXI/FXIaantibodies, e.g., NOV1090 and NOV1401. In another aspect, the presentinvention provides FXIa binding antibodies that comprise the heavy chainand light chain CDR1s, CDR2s, and CDR3s as described in Table 1, orcombinations thereof. The amino acid sequences of the VH CDR1s of theantibodies are shown in SEQ ID NOs: 3 and 23. The amino acid sequencesof the VH CDR2s of the antibodies and are shown in SEQ ID NOs: 4 and 24.The amino acid sequences of the VH CDR3s of the antibodies are shown inSEQ ID NOs: 5 and 25. The amino acid sequences of the VL CDR1s of theantibodies are shown in SEQ ID NOs: 13 and 33. The amino acid sequencesof the VL CDR2s of the antibodies are shown in SEQ ID NOs: 14 and 34.The amino acid sequences of the VL CDR3s of the antibodies are shown inSEQ ID NOs: 15 and 35. These CDR regions are delineated using the Kabatsystem.

Alternatively, as defined using the Chothia system (Al-Lazikani et al.,(1997) JMB 273,927-948), the amino acid sequences of the VH CDR1s of theantibodies are shown in SEQ ID NOs: 6 and 26. The amino acid sequencesof the VH CDR2s of the antibodies and are shown in SEQ ID NOs: 7 and 27.The amino acid sequences of the VH CDR3s of the antibodies are shown inSEQ ID NOs: 8 and 28. The amino acid sequences of the VL CDR1s of theantibodies are shown in SEQ ID NOs: 16 and 36. The amino acid sequencesof the VL CDR2s of the antibodies are shown in SEQ ID NOs: 17 and 37.The amino acid sequences of the VL CDR3s of the antibodies are shown inSEQ ID NOs: 18 and 38.

Alternatively, as defined using the Combined system, the amino acidsequences of the VH CDR1 of the antibodies are shown in SEQ ID NO: 46.The amino acid sequences of the VH CDR2 of the antibodies and are shownin SEQ ID NO: 4. The amino acid sequences of the VH CDR3 of theantibodies are shown in SEQ ID NO: 5. The amino acid sequences of the VLCDR1 of the antibodies are shown in SEQ ID NO: 33. The amino acidsequences of the VL CDR2 of the antibodies are shown in SEQ ID NO: 14.The amino acid sequences of the VL CDR3 of the antibodies are shown inSEQ ID NO: 15.

Alternatively, as defined using the IMGT numbering scheme, the aminoacid sequences of the VH CDR1 of the antibodies are shown in SEQ ID NO:43. The amino acid sequences of the VH CDR2 of the antibodies and areshown in SEQ ID NO: 44. The amino acid sequences of the VH CDR3 of theantibodies are shown in SEQ ID NO: 45. The amino acid sequences of theVL CDR1 of the antibodies are shown in SEQ ID NO: 47. The amino acidsequences of the VL CDR2 of the antibodies are shown in SEQ ID NO: 37.The amino acid sequences of the VL CDR3 of the antibodies are shown inSEQ ID NO: 15.

Given that each of these antibodies can bind to FXI and/or FXIa and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequencescan be “mixed and matched” (i.e., CDRs from different antibodies can bemixed and matched, although each antibody preferably contains a VH CDR1,2 and 3 and a VL CDR1, 2 and 3 to create other FXI and/or FXIa bindingmolecules of the invention. Such “mixed and matched” FXI and/or FXIabinding antibodies can be tested using the binding assays known in theart and those described in the Examples (e.g., ELISAs, SET, BIACORE™assays). When VH CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular VH sequence should be replacedwith a structurally similar CDR sequence(s). Likewise, when VL CDRsequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular VL sequence should be replaced with a structurallysimilar CDR sequence(s). It will be readily apparent to the ordinarilyskilled artisan that novel VH and VL sequences can be created bysubstituting one or more VH and/or VL CDR region sequences withstructurally similar sequences from the CDR sequences shown herein formonoclonal antibodies of the present invention. In addition to theforegoing, in one embodiment, the antigen binding fragments of theantibodies described herein can comprise a VH CDR1, 2, and 3, or a VLCDR 1, 2, and 3, wherein the fragment binds to FXI and/or FXIa as asingle variable domain.

In certain embodiments of the invention, the antibodies or antigenbinding fragments thereof may have the heavy and light chain sequencesof the Fabs described in Table 1. More specifically, the antibody orantigen binding fragments thereof may have the heavy and light sequenceof NOV1090 and NOV1401.

In other embodiments of the invention the antibody or antigen bindingfragment in that specifically binds FXI and/or FXIa comprises a heavychain variable region CDR1, a heavy chain variable region CDR2, a heavychain variable region CDR3, a light chain variable region CDR1, a lightchain variable region CDR2, and a light chain variable region CDR3 asdefined by Kabat and described in Table 1. In still other embodiments ofthe invention the antibody or antigen binding fragment in thatspecifically binds FXI and/or FXIa comprises a heavy chain variableregion CDR1, a heavy chain variable region CDR2, a heavy chain variableregion CDR3, a light chain variable region CDR1, a light chain variableregion CDR2, and a light chain variable region CDR3 as defined byChothia and described in Table 1. In other embodiments, the antibody orantigen binding fragment in that specifically binds FXI and/or FXIacomprises a heavy chain variable region CDR1, a heavy chain variableregion CDR2, a heavy chain variable region CDR3, a light chain variableregion CDR1, a light chain variable region CDR2, and a light chainvariable region CDR3 as defined by the Combined system and described inTable 1. In still other embodiments of the invention the antibody orantigen binding fragment in that specifically binds FXI and/or FXIacomprises a heavy chain variable region CDR1, a heavy chain variableregion CDR2, a heavy chain variable region CDR3, a light chain variableregion CDR1, a light chain variable region CDR2, and a light chainvariable region CDR3 as defined by IMGT and described in Table 1.

In a specific embodiment, the invention includes an antibody thatspecifically binds to FXI and/or FXIa comprising a heavy chain variableregion CDR1 of SEQ ID NO: 3; a heavy chain variable region CDR2 of SEQID NO: 4; a heavy chain variable region CDR3 of SEQ ID NO: 5; a lightchain variable region CDR1 of SEQ ID NO: 13; a light chain variableregion CDR2 of SEQ ID NO: 14; and a light chain variable region CDR3 ofSEQ ID NO: 15.

In a specific embodiment, the invention includes an antibody thatspecifically binds to FXI and/or FXIa comprising a heavy chain variableregion CDR1 of SEQ ID NO: 23; a heavy chain variable region CDR2 of SEQID NO: 24; a heavy chain variable region CDR3 of SEQ ID NO: 25; a lightchain variable region CDR1 of SEQ ID NO: 33; a light chain variableregion CDR2 of SEQ ID NO: 34; and a light chain variable region CDR3 ofSEQ ID NO: 35.

In a specific embodiment, the invention includes an antibody thatspecifically binds to FXI and/or FXIa comprising a heavy chain variableregion CDR1 of SEQ ID NO: 6; a heavy chain variable region CDR2 of SEQID NO: 7; a heavy chain variable region CDR3 of SEQ ID NO: 8; a lightchain variable region CDR1 of SEQ ID NO: 16; a light chain variableregion CDR2 of SEQ ID NO: 17; and a light chain variable region CDR3 ofSEQ ID NO: 18.

In a specific embodiment, the invention includes an antibody thatspecifically binds to FXI and/or FXIa comprising a heavy chain variableregion CDR1 of SEQ ID NO: 26; a heavy chain variable region CDR2 of SEQID NO: 27; a heavy chain variable region CDR3 of SEQ ID NO: 28; a lightchain variable region CDR1 of SEQ ID NO: 36; a light chain variableregion CDR2 of SEQ ID NO: 37; and a light chain variable region CDR3 ofSEQ ID NO: 38.

In a specific embodiment, provided herein is an antibody thatspecifically binds to FXI and/or FXIa comprising a heavy chain variableregion CDR1 of SEQ ID NO: 43; a heavy chain variable region CDR2 of SEQID NO: 44; a heavy chain variable region CDR3 of SEQ ID NO: 45; a lightchain variable region CDR1 of SEQ ID NO: 47; a light chain variableregion CDR2 of SEQ ID NO: 37 and a light chain variable region CDR3 ofSEQ ID NO: 15.

In a specific embodiment, provided herein is an antibody thatspecifically binds to FXI and/or FXIa comprising a heavy chain variableregion CDR1 of SEQ ID NO: 46; a heavy chain variable region CDR2 of SEQID NO: 4; a heavy chain variable region CDR3 of SEQ ID NO: 5; a lightchain variable region CDR1 of SEQ ID NO: 33; a light chain variableregion CDR2 of SEQ ID NO: 14 and a light chain variable region CDR3 ofSEQ ID NO: 15.

In certain embodiments, the invention includes antibodies or antigenbinding fragments that specifically bind to FXI and/or FXIa as describedin Table 1. In a preferred embodiment, the antibody, or antigen bindingfragment, that binds FXI and/or FXIa is NOV1090 and NOV1401.

As used herein, a human antibody comprises heavy or light chain variableregions or full length heavy or light chains that are “the product of”or “derived from” a particular germline sequence if the variable regionsor full length chains of the antibody are obtained from a system thatuses human germline immunoglobulin genes. Such systems includeimmunizing a transgenic mouse carrying human immunoglobulin genes withthe antigen of interest or screening a human immunoglobulin gene librarydisplayed on phage with the antigen of interest. A human antibody thatis “the product of” or “derived from” a human germline immunoglobulinsequence can be identified as such by comparing the amino acid sequenceof the human antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody.

A human antibody that is “the product of” or “derived from” a particularhuman germline immunoglobulin sequence may contain amino aciddifferences as compared to the germline sequence, due to, for example,naturally occurring somatic mutations or intentional introduction ofsite-directed mutations. However, in the VH or VL framework regions, aselected human antibody typically is at least 90% identical in aminoacids sequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least60%, 70%, 80%, 90%, or at least 95%, or even at least 96%, 97%, 98%, or99% identical in amino acid sequence to the amino acid sequence encodedby the germline immunoglobulin gene.

Typically, a recombinant human antibody will display no more than 10amino acid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene in the VH or VL framework regions. Incertain cases, the human antibody may display no more than 5, or even nomore than 4, 3, 2, or 1 amino acid difference from the amino acidsequence encoded by the germline immunoglobulin gene. Examples of humangermline immunoglobulin genes include, but are not limited to thevariable domain germline fragments described below, as well as DP47 andDPK9.

Homologous Antibodies

In yet another embodiment, the present invention provides an antibody,or an antigen binding fragment thereof, comprising amino acid sequencesthat are homologous to the sequences described in Table 1 (e.g., SEQ IDNOs: 29, 31, 39, or 41), and the antibody binds to an FXI and/or FXIaprotein (e.g., human, rabbit, and cynomolgus monkey FXIa), and retainsthe desired functional properties of those antibodies described in Table1 such as NOV1090 and NOV1401. In specific aspects, such homologousantibodies retain the CDR amino acid sequences described in Table 1(e.g., Kabat CDRs, Chothia CDRs, IMGT CDRs, or Combined CDRs).

For example, the invention provides an isolated antibody, or afunctional antigen binding fragment thereof, comprising a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises an amino acid sequence that is at least80%, at least 90%, or at least 95% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9 and 29; the lightchain variable domain comprises an amino acid sequence that is at least80%, at least 90%, or at least 95% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19 and 39; and theantibody specifically binds to FXI and/or FXIa (e.g., human, rabbit, andcynomolgus monkey FXIa). In one embodiment, an isolated antibody, or afunctional antigen binding fragment thereof, comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises an amino acid sequence that is at least80%, at least 90%, or at least 95% identical to the amino acid sequenceof SEQ ID NO: 9; the light chain variable domain comprises an amino acidsequence that is at least 80%, at least 90%, or at least 95% identicalto the amino acid sequence of SEQ ID NO: 19; and the antibodyspecifically binds to FXI and/or FXIa (e.g., human, rabbit, andcynomolgus monkey FXIa). In one embodiment, an isolated antibody, or afunctional antigen binding fragment thereof, comprises a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises an amino acid sequence that is at least80%, at least 90%, or at least 95% identical to the amino acid sequenceof SEQ ID NO: 29; the light chain variable domain comprises an aminoacid sequence that is at least 80%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 39; and the antibodyspecifically binds to FXI and/or FXIa (e.g., human, rabbit, andcynomolgus monkey FXIa). In certain aspects of the invention the heavyand light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 sequences as defined by Kabat, for example SEQ ID NOs:3, 4, 5, 13, 14, and 15, respectively. In certain other aspects of theinvention the heavy and light chain sequences further comprise HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Chothia,for example SEQ ID NOs: 6, 7, 8, 16, 17, and 18, respectively. Incertain other aspects, the heavy and light chain sequences furthercomprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences asdefined by the Combined system, for example SEQ ID NOs: 46, 4, 5, 33,14, and 15, respectively. In certain other aspects, the heavy and lightchain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, andLCDR3 sequences as defined by IMGT, for example SEQ ID NOs: 43, 44, 45,47, 37, and 15, respectively.

In other embodiments, the VH and/or VL amino acid sequences may be 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequencesset forth in Table 1. In other embodiments, the VH and/or VL amino acidsequences may be identical except for an amino acid substitution in nomore than 1, 2, 3, 4 or 5 amino acid positions. An antibody having VHand VL regions having high (i.e., 80% or greater) identity to the VH andVL regions of those described in Table 1 can be obtained by mutagenesis(e.g., site-directed or PCR-mediated mutagenesis) of nucleic acidmolecules encoding SEQ ID NOs: 10 or 30 and SEQ ID NOs: 20 and 40,respectively, followed by testing of the encoded altered antibody forretained function using the functional assays described herein.

In other embodiments, the full length heavy chain and/or full lengthlight chain amino acid sequences may be 50% 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to the sequences set forth in Table 1(e.g., SEQ ID NOs: 11 and/or 21, or 31 and/or 41). An antibody having afull length heavy chain and full length light chain having high (i.e.,80% or greater) identity to the full length heavy chains of any of SEQID NOs: 11 or 31, and full length light chains of any of SEQ ID NOs: 21or 41, can be obtained by mutagenesis (e.g., site-directed orPCR-mediated mutagenesis) of nucleic acid molecules encoding suchpolypeptides, followed by testing of the encoded altered antibody forretained function using the functional assays described herein.

In one aspect, provided herein is an isolated antibody, or a functionalantigen binding fragment thereof, comprising a heavy chain and a lightchain, wherein the heavy chain comprises an amino acid sequence that isat least 80%, at least 90%, or at least 95% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 11 and 31;the light chain comprises an amino acid sequence that is at least 80%,at least 90%, or at least 95% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21 and 41; and theantibody specifically binds to FXI and/or FXIa (e.g., human, rabbit, andcynomolgus monkey FXIa). In one embodiment, an isolated antibody, or afunctional antigen binding fragment thereof, comprises a heavy chain anda light chain, wherein the heavy chain comprises an amino acid sequencethat is at least 80%, at least 90%, or at least 95% identical to theamino acid sequence of SEQ ID NO: 11; the light chain comprises an aminoacid sequence that is at least 80%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 21; and the antibodyspecifically binds to FXI and/or FXIa (e.g., human, rabbit, andcynomolgus monkey FXIa). In one embodiment, an isolated antibody, or afunctional antigen binding fragment thereof, comprises a heavy chain anda light chain, wherein the heavy chain comprises an amino acid sequencethat is at least 80%, at least 90%, or at least 95% identical to theamino acid sequence of SEQ ID NO: 31; the light chain comprises an aminoacid sequence that is at least 80%, at least 90%, or at least 95%identical to the amino acid sequence of SEQ ID NO: 41; and the antibodyspecifically binds to FXI and/or FXIa (e.g., human, rabbit, andcynomolgus monkey FXIa). In certain aspects of the invention the heavyand light chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 sequences as defined by Kabat, for example SEQ ID NOs:3, 4, 5, 13, 14, and 15, respectively. In certain other aspects of theinvention the heavy and light chain sequences further comprise HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences as defined by Chothia,for example SEQ ID NOs: 6, 7, 8, 16, 17, and 18, respectively. Incertain other aspects, the heavy and light chain sequences furthercomprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences asdefined by the Combined system, for example SEQ ID NOs: 46, 4, 5, 33,14, and 15, respectively. In certain other aspects, the heavy and lightchain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, andLCDR3 sequences as defined by IMGT, for example SEQ ID NOs: 43, 44, 45,47, 37, and 15, respectively.

In other embodiments, the full length heavy chain and/or full lengthlight chain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% identical to the sequences set forth in Table 1 (e.g.,SEQ ID NOs: 12 and/or 22, or 32 and/or 42).

In other embodiments, the variable regions of heavy chain and/or thevariable regions of light chain nucleotide sequences may be 60%, 70%,80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forthin Table 1 (e.g., SEQ ID NOs: 10 and/or 20, or 30 and/or 40).

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity equals number of identical positions/total number ofpositions×100), taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.For example, such searches can be performed using the BLAST program(version 2.0) of Altschul, et al., 1990 J. Mol. Biol. 215:403-10.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention has a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences, whereinone or more of these CDR sequences have specified amino acid sequencesbased on the antibodies described herein or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the FXIa-binding antibodies of the invention.

Accordingly, the invention provides an isolated antibody, or an antigenbinding fragment thereof, consisting of a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 sequences and a light chain variableregion comprising CDR1, CDR2, and CDR3 sequences, wherein: the heavychain variable region CDR1 amino acid sequences are selected from thegroup consisting of SEQ ID NOs: 3 and 23, and conservative modificationsthereof; the heavy chain variable region CDR2 amino acid sequences areselected from the group consisting of SEQ ID NOs: 4 and 24, andconservative modifications thereof; the heavy chain variable region CDR3amino acid sequences are selected from the group consisting of SEQ IDNOs: 5 and 25, and conservative modifications thereof; the light chainvariable regions CDR1 amino acid sequences are selected from the groupconsisting of SEQ ID NOs: 13 and 33, and conservative modificationsthereof; the light chain variable regions CDR2 amino acid sequences areselected from the group consisting of SEQ ID NOs: 14 and 34, andconservative modifications thereof; the light chain variable regions ofCDR3 amino acid sequences are selected from the group consisting of SEQID NOs: 15 and 35, and conservative modifications thereof; and theantibody or antigen binding fragments thereof specifically binds toFXIa.

In one aspect, provided herein is an isolated antibody, or an antigenbinding fragment thereof, consisting of a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 sequences and a light chain variableregion comprising CDR1, CDR2, and CDR3 sequences, wherein: the heavychain variable region CDR1 amino acid sequences are selected from thegroup consisting of those described in Table 1, and conservativemodifications thereof; the heavy chain variable region CDR2 amino acidsequences are selected from the group consisting of those described inTable 1, and conservative modifications thereof; the heavy chainvariable region CDR3 amino acid sequences are selected from the groupconsisting of those described in Table 1, and conservative modificationsthereof; the light chain variable regions CDR1 amino acid sequences areselected from the group consisting of those described in Table 1, andconservative modifications thereof; the light chain variable regionsCDR2 amino acid sequences are selected from the group consisting ofthose described in Table 1, and conservative modifications thereof; thelight chain variable regions of CDR3 amino acid sequences are selectedfrom the group consisting of those described in Table 1, andconservative modifications thereof; and the antibody or antigen bindingfragments thereof specifically binds to FXIa.

In other embodiments, the antibody of the invention is optimized forexpression in a mammalian cell has a full length heavy chain sequenceand a full length light chain sequence, wherein one or more of thesesequences have specified amino acid sequences based on the antibodiesdescribed herein or conservative modifications thereof, and wherein theantibodies retain the desired functional properties of the FXIa bindingantibodies of the invention. Accordingly, the invention provides anisolated antibody optimized for expression in a mammalian cellconsisting of a full length heavy chain and a full length light chainwherein the full length heavy chain has amino acid sequences selectedfrom the group of SEQ ID NOs: 11 or 31, and conservative modificationsthereof; and the full length light chain has amino acid sequencesselected from the group of SEQ ID NOs: 21 or 41, and conservativemodifications thereof; and the antibody specifically binds to FXI and/orFXIa (e.g., human, rabbit, and cynomolgus monkey FXIa).

Antibodies that Bind to the Same Epitope

The present invention provides antibodies that bind to the same epitopeas the FXI and/or FXIa binding antibodies described in Table 1.Additional antibodies can therefore be identified based on their abilityto compete (e.g., to competitively inhibit the binding of, in astatistically significant manner, by binding to the same or overlappingepitope) with other antibodies of the invention in FXI and/or FXIabinding assays (such as those described in the Examples Section). Theability of a test antibody to inhibit the binding of antibodies of thepresent invention to a FXI and/or FXIa protein demonstrates that thetest antibody can compete with that antibody for binding to FXI and/orFXIa; such an antibody may, according to non-limiting theory, bind tothe same or a related (e.g., a structurally similar or spatiallyproximal) epitope on the FXI and/or FXIa protein as the antibody withwhich it competes. In a certain embodiment, the antibody that binds tothe same epitope on FXI and/or FXIa as the antibodies of the presentinvention is a human monoclonal antibody. Such human monoclonalantibodies can be prepared and isolated as described herein.

As used herein, an antibody “competes” for binding when the competingantibody binds to the same FXI and/or FXIa epitope as an antibody orantigen binding fragment of the invention (e.g., NOV1401 or NOV1090) andinhibits FXI and/or FXIa binding of an antibody or antigen bindingfragment of the invention by more than 50% (for example, 80%, 85%, 90%,95%, 98% or 99%) in the presence of an equimolar concentration ofcompeting antibody. This may be determined, for instance, in acompetitive binding assay, by any of the methods well known to those ofskill in the art.

As used herein, an antibody or antigen binding fragment thereof does not“compete” with an FXI and/or FXIa antibody or antigen binding fragmentof the invention (e.g., NOV1401 or NOV1090) unless said competingantibody or antigen binding fragment thereof binds the same FXI and/orFXIa epitope, or an overlapping FXI and/or FXIa epitope, as an antibodyor antigen binding fragment of the invention. As used herein, acompeting antibody or antigen binding fragment thereof does not includeone which (i) sterically blocks an antibody or antigen binding fragmentof the invention from binding its target (e.g., if said competingantibody binds to a nearby, non-overlapping FXI and/or FXIa epitope andphysically prevents an antibody or antigen binding fragment of theinvention from binding its target); and/or (ii) binds to a different,non-overlapping FXI and/or FXIa epitope and induces a conformationalchange to the FXI and/or FXIa protein such that said protein can nolonger be bound by an FXI and/or FXIa antibody or antigen bindingfragment of the invention in a way that would occur absent saidconformational change.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the VH and/or VL sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i. e., VH and/or VL), for example within oneor more CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad., U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.)

Accordingly, another embodiment of the invention pertains to an isolatedantibody, or an antigen binding fragment thereof, comprising a heavychain variable region comprising CDR1 sequences having an amino acidsequence selected from the group consisting of SEQ ID NOs: 3 and 23;CDR2 sequences having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4 and 24; CDR3 sequences having an amino acidsequence selected from the group consisting of SEQ ID NOs: 5 and 25,respectively; and a light chain variable region having CDR1 sequenceshaving an amino acid sequence selected from the group consisting of SEQID NOs: 13 and 33; CDR2 sequences having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 14 and 34; and CDR3 sequencesconsisting of an amino acid sequence selected from the group consistingof SEQ ID NOs: 15 and 35, respectively. Thus, such antibodies containthe VH and VL CDR sequences of monoclonal antibodies, yet may containdifferent framework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the world wide web at mrc-cpe.cam.ac.uk/vbase),as well as in Kabat, E. A., et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.,1992 J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. JImmunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

An example of framework sequences for use in the antibodies of theinvention are those that are structurally similar to the frameworksequences used by selected antibodies of the invention, e.g., consensussequences and/or framework sequences used by monoclonal antibodies ofthe invention. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3sequences, can be grafted onto framework regions that have the identicalsequence as that found in the germline immunoglobulin gene from whichthe framework sequence derive, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al). Frameworks that can be utilized as scaffoldson which to build the antibodies and antigen binding fragments describedherein include, but are not limited to VH1A, VH1B, VH3, Vk1, Vl2, andVk2. Additional frameworks are known in the art and may be found, forexample, in the vBase data base on the world wide web atvbase.mrc-cpe.cam.ac.uk/index.php?&MMN_position=1:1.

Accordingly, an embodiment of the invention relates to isolated FXIabinding antibodies, or antigen binding fragments thereof, comprising aheavy chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 9 and 29, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions in the framework region of such sequences, andfurther comprising a light chain variable region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 19 or 39, oran amino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions in the framework region of suchsequences.

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples Section. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Accordingly, in another embodiment, the invention provides isolatedFXIa-binding antibodies, or antigen binding fragments thereof,consisting of a heavy chain variable region having a VH CDR1 regionconsisting of an amino acid sequence selected from the group having SEQID NOs: 3 and 23 or an amino acid sequence having one, two, three, fouror five amino acid substitutions, deletions or additions as compared toSEQ ID NOs: 3 and 23; a VH CDR2 region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4 and 24 or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 4 and24; a VH CDR3 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 5 and 25, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 5 and 25; a VL CDR1 regionhaving an amino acid sequence selected from the group consisting of SEQID NOs: 13 and 33, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 13 and 33; a VL CDR2 region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 14 and 34, oran amino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 14 and34; and a VL CDR3 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 15 and 35, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 15 and 35.

Accordingly, in another embodiment, the invention provides isolatedFXIa-binding antibodies, or antigen binding fragments thereof,consisting of a heavy chain variable region having a VH CDR1 regionconsisting of an amino acid sequence selected from the group having SEQID NOs: 6 and 26 or an amino acid sequence having one, two, three, fouror five amino acid substitutions, deletions or additions as compared toSEQ ID NOs: 6 and 26; a VH CDR2 region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 7 and 27 or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 7 and27; a VH CDR3 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 8 and 28, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 8 and 28; a VL CDR1 regionhaving an amino acid sequence selected from the group consisting of SEQID NOs: 16 and 36, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 16 and 36; a VL CDR2 region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 17 and 37, oran amino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 17 and37; and a VL CDR3 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 18 and 38, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 18 and 38.

Grafting Antigen-Binding Domains into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which specifically binds to FXIa. Such frameworks orscaffolds include the 5 main idiotypes of human immunoglobulins, orfragments thereof, and include immunoglobulins of other animal species,preferably having humanized aspects. Single heavy-chain antibodies suchas those identified in camelids are of particular interest in thisregard. Novel frameworks, scaffolds and fragments continue to bediscovered and developed by those skilled in the art.

In one aspect, the invention pertains to generating non-immunoglobulinbased antibodies using non-immunoglobulin scaffolds onto which CDRs ofthe invention can be grafted. Known or future non-immunoglobulinframeworks and scaffolds may be employed, as long as they comprise abinding region specific for the target FXI and/or FXIa protein. Knownnon-immunoglobulin frameworks or scaffolds include, but are not limitedto, fibronectin (Compound Therapeutics, Inc., Waltham, Mass.), ankyrin(Molecular Partners AG, Zurich, Switzerland), domain antibodies(Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium),lipocalin (Pieris Proteolab AG, Freising, Germany), small modularimmuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.),maxybodies (Avidia, Inc., Mountain View, Calif.), Protein A (AffibodyAG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil ProteinsGmbH, Halle, Germany).

The fibronectin scaffolds are based on fibronectin type III domain(e.g., the tenth module of the fibronectin type III (10 Fn3 domain)).The fibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (see U.S. Pat. No.6,818,418). These fibronectin-based scaffolds are not an immunoglobulin,although the overall fold is closely related to that of the smallestfunctional antibody fragment, the variable region of the heavy chain,which comprises the entire antigen recognition unit in camel and llamaIgG. Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the invention using standardcloning techniques.

The ankyrin technology is based on using proteins with ankyrin derivedrepeat modules as scaffolds for bearing variable regions which can beused for binding to different targets. The ankyrin repeat module is a 33amino acid polypeptide consisting of two anti-parallel α-helices and aβ-turn. Binding of the variable regions is mostly optimized by usingribosome display.

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example, U.S.Patent Application Publication Nos. 20040175756; 20050053973;20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate affibody libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibodymolecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of affibody molecules issimilar to that of an antibody.

Anticalins are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids. The proteinarchitecture is reminiscent of immunoglobulins, with hypervariable loopson top of a rigid framework. However, in contrast with antibodies ortheir recombinant fragments, lipocalins are composed of a singlepolypeptide chain with 160 to 180 amino acid residues, being justmarginally bigger than a single immunoglobulin domain. The set of fourloops, which makes up the binding pocket, shows pronounced structuralplasticity and tolerates a variety of side chains. The binding site canthus be reshaped in a proprietary process in order to recognizeprescribed target molecules of different shape with high affinity andspecificity. One protein of lipocalin family, the bilin-binding protein(BBP) of Pieris Brassicae has been used to develop anticalins bymutagenizing the set of four loops. One example of a patent applicationdescribing anticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New affilin molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.Affilin molecules do not show any structural homology to immunoglobulinproteins. Currently, two affilin scaffolds are employed, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368.

Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-likemolecules (MW 1-2 kDa) mimicking beta-hairpin secondary structures ofproteins, the major secondary structure involved in protein-proteininteractions.

The present invention provides fully human antibodies that specificallybind to a FXIa protein. Compared to the chimeric or humanizedantibodies, the human FXIa-binding antibodies of the invention havefurther reduced antigenicity when administered to human subjects.

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Calelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as VHH can be obtained by genetic engineering to yielda small protein having high affinity for a target, resulting in a lowmolecular weight antibody-derived protein known as a “camelid nanobody”.See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B.et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; andLauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries ofcamelid antibodies and antibody fragments are commercially available,for example, from Ablynx, Ghent, Belgium. As with other antibodies ofnon-human origin, an amino acid sequence of a camelid antibody can bealtered recombinantly to obtain a sequence that more closely resembles ahuman sequence, i.e., the nanobody can be “humanized”. Thus the naturallow antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitate drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present invention is a camelid antibody ornanobody having high affinity for FXI and/or FXIa. In certainembodiments herein, the camelid antibody or nanobody is naturallyproduced in the camelid animal, i.e., is produced by the camelidfollowing immunization with FXI and/or FXIa or a peptide fragmentthereof, using techniques described herein for other antibodies.Alternatively, the FXI and/or FXIa-binding camelid nanobody isengineered, i.e., produced by selection for example from a library ofphage displaying appropriately mutagenized camelid nanobody proteinsusing panning procedures with FXI and/or FXIa, and/or domains and/orpeptide fragments thereof, as a target as described in the examplesherein. Engineered nanobodies can further be customized by geneticengineering to have a half-life in a recipient subject of from 45minutes to two weeks. In a specific embodiment, the camelid antibody ornanobody is obtained by grafting the CDRs sequences of the heavy orlight chain of the human antibodies of the invention into nanobody orsingle domain antibody framework sequences, as described for example inPCT/EP93/02214.

Bispecific Molecules and Multivalent Antibodies

In another aspect, the present invention features bispecific ormultispecific molecules comprising a FXI and/or FXIa-binding antibody,or a fragment thereof, of the invention. An antibody of the invention,or antigen-binding regions thereof, can be derivatized or linked toanother functional molecule, e.g., another peptide or protein (e.g.,another antibody or ligand for a receptor) to generate a bispecificmolecule that binds to at least two different binding sites or targetmolecules. The antibody of the invention may in fact be derivatized orlinked to more than one other functional molecule to generatemulti-specific molecules that bind to more than two different bindingsites and/or target molecules; such multi-specific molecules are alsointended to be encompassed by the term “bispecific molecule” as usedherein. To create a bispecific molecule of the invention, an antibody ofthe invention can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother binding molecules, such as another antibody, antibody fragment,peptide or binding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for FXI and/or FXIaand a second binding specificity for a second target epitope. Forexample, the second target epitope is another epitope of FXI and/or FXIadifferent from the first target epitope.

Additionally, for the invention in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., a Fab, Fab′, F(ab′)2, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778.

Diabodies are bivalent, bispecific molecules in which VH and VL domainsare expressed on a single polypeptide chain, connected by a linker thatis too short to allow for pairing between the two domains on the samechain. The VH and VL domains pair with complementary domains of anotherchain, thereby creating two antigen binding sites (see e.g., Holliger etal., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994Structure 2:1121-1123). Diabodies can be produced by expressing twopolypeptide chains with either the structure VHA-VLB and VHB-VLA (VH-VLconfiguration), or VLA-VHB and VLB-VHA (VL-VH configuration) within thesame cell. Most of them can be expressed in soluble form in bacteria.Single chain diabodies (scDb) are produced by connecting the twodiabody-forming polypeptide chains with linker of approximately 15 aminoacid residues (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45(3-4):128-30; Wu et al., 1996 Immunotechnology,2(1):21-36). scDb can be expressed in bacteria in soluble, activemonomeric form (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45(34): 128-30; Wu et al., 1996 Immunotechnology,2(1):21-36; Pluckthun and Pack, 1997 Immunotechnology, 3(2): 83-105;Ridgway et al., 1996 Protein Eng., 9(7):617-21). A diabody can be fusedto Fc to generate a “di-diabody” (see Lu et al., 2004 J. Biol. Chem.,279(4):2856-65).

Other antibodies which can be employed in the bispecific molecules ofthe invention are murine, chimeric and humanized monoclonal antibodies.

Bispecific molecules can be prepared by conjugating the constituentbinding specificities, using methods known in the art. For example, eachbinding specificity of the bispecific molecule can be generatedseparately and then conjugated to one another. When the bindingspecificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-I-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No.78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb x mAb, mAb xFab, Fab x F(ab′)2 or ligand x Fab fusion protein. A bispecific moleculeof the invention can be a single chain molecule comprising one singlechain antibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

In another aspect, the present invention provides multivalent compoundscomprising at least two identical or different antigen-binding portionsof the antibodies of the invention binding to FXIa. The antigen-bindingportions can be linked together via protein fusion or covalent ornon-covalent linkage. Alternatively, methods of linkage have beendescribed for the bispecfic molecules. Tetravalent compounds can beobtained for example by cross-linking antibodies of the antibodies ofthe invention with an antibody that binds to the constant regions of theantibodies of the invention, for example the Fc or hinge region.

Trimerizing domain are described for example in Borean patent EP 1 01228061. Pentamerizing modules are described for example inPCT/EP97/05897.

Antibodies with Extended Half Life

The present invention provides for antibodies that specifically bind toFXIa protein which have an extended half-life in vivo.

Many factors may affect a protein's half-life in vivo. For examples,kidney filtration, metabolism in the liver, degradation by proteolyticenzymes (proteases), and immunogenic responses (e.g., proteinneutralization by antibodies and uptake by macrophages and dendriticcells). A variety of strategies can be used to extend the half-life ofthe antibodies of the present invention. For example, by chemicallinkage to polyethyleneglycol (PEG), reCODE PEG, antibody scaffold,polysialic acid (PSA), hydroxyethyl starch (HES), albumin-bindingligands, and carbohydrate shields; by genetic fusion to proteins bindingto serum proteins, such as albumin, IgG, FcRn, and transferring; bycoupling (genetically or chemically) to other binding moieties that bindto serum proteins, such as nanobodies, Fabs, DARPins, avimers,affibodies, and anticalins; by genetic fusion to rPEG, albumin, domainof albumin, albumin-binding proteins, and Fc; or by incorporation intonanocarriers, slow release formulations, or medical devices.

To prolong the serum circulation of antibodies in vivo, inert polymermolecules such as high molecular weight PEG can be attached to theantibodies or a fragment thereof with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of the antibodies or via epsilon-amino groups present onlysine residues. To pegylate an antibody, the antibody, or fragmentthereof, typically is reacted with polyethylene glycol (PEG), such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. The pegylation can be carried out by an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Linear or branched polymer derivatization that results in minimal lossof biological activity will be used. The degree of conjugation can beclosely monitored by SDS-PAGE and mass spectrometry to ensure properconjugation of PEG molecules to the antibodies. Unreacted PEG can beseparated from antibody-PEG conjugates by size-exclusion or byion-exchange chromatography. PEG-derivatized antibodies can be testedfor binding activity as well as for in vivo efficacy using methodswell-known to those of skill in the art, for example, by immunoassaysdescribed herein. Methods for pegylating proteins are known in the artand can be applied to the antibodies of the invention. See for example,EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Other modified pegylation technologies include reconstituting chemicallyorthogonal directed engineering technology (ReCODE PEG), whichincorporates chemically specified side chains into biosynthetic proteinsvia a reconstituted system that includes tRNA synthetase and tRNA. Thistechnology enables incorporation of more than 30 new amino acids intobiosynthetic proteins in E. coli, yeast, and mammalian cells. The tRNAincorporates a nonnative amino acid any place an amber codon ispositioned, converting the amber from a stop codon to one that signalsincorporation of the chemically specified amino acid.

Recombinant pegylation technology (rPEG) can also be used for serumhalflife extension. This technology involves genetically fusing a300-600 amino acid unstructured protein tail to an existingpharmaceutical protein. Because the apparent molecular weight of such anunstructured protein chain is about 15-fold larger than its actualmolecular weight, the serum half-life of the protein is greatlyincreased. In contrast to traditional PEGylation, which requireschemical conjugation and repurification, the manufacturing process isgreatly simplified and the product is homogeneous.

Polysialytion is another technology, which uses the natural polymerpolysialic acid (PSA) to prolong the active life and improve thestability of therapeutic peptides and proteins. PSA is a polymer ofsialic acid (a sugar). When used for protein and therapeutic peptidedrug delivery, polysialic acid provides a protective microenvironment onconjugation. This increases the active life of the therapeutic proteinin the circulation and prevents it from being recognized by the immunesystem. The PSA polymer is naturally found in the human body. It wasadopted by certain bacteria which evolved over millions of years to coattheir walls with it. These naturally polysialylated bacteria were thenable, by virtue of molecular mimicry, to foil the body's defense system.PSA, nature's ultimate stealth technology, can be easily produced fromsuch bacteria in large quantities and with predetermined physicalcharacteristics. Bacterial PSA is completely non-immunogenic, even whencoupled to proteins, as it is chemically identical to PSA in the humanbody.

Another technology includes the use of hydroxyethyl starch (“HES”)derivatives linked to antibodies. HES is a modified natural polymerderived from waxy maize starch and can be metabolized by the body'senzymes. HES solutions are usually administered to substitute deficientblood volume and to improve the rheological properties of the blood.Hesylation of an antibody enables the prolongation of the circulationhalf-life by increasing the stability of the molecule, as well as byreducing renal clearance, resulting in an increased biological activity.By varying different parameters, such as the molecular weight of HES, awide range of HES antibody conjugates can be customized.

Antibodies having an increased half-life in vivo can also be generatedintroducing one or more amino acid modifications (i.e., substitutions,insertions or deletions) into an IgG constant domain, or FcRn bindingfragment thereof (preferably a Fc or hinge Fc domain fragment). See,e.g., International Publication No. WO 98/23289; InternationalPublication No. WO 97/34631; and U.S. Pat. No. 6,277,375.

Further, antibodies can be conjugated to albumin (e.g., human serumalbumin; HSA) in order to make the antibody or antibody fragment morestable in vivo or have a longer half life in vivo. The techniques arewell-known in the art, see, e.g., International Publication Nos. WO93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP413,622. In addition, in the context of a bispecific antibody asdescribed above, the specificities of the antibody can be designed suchthat one binding domain of the antibody binds to FXIa while a secondbinding domain of the antibody binds to serum albumin, preferably HSA.

The strategies for increasing half-life is especially useful innanobodies, fibronectin-based binders, and other antibodies or proteinsfor which increased in vivo half-life is desired.

Antibody Conjugates

The present invention provides antibodies or fragments thereof thatspecifically bind to a FXIa protein recombinantly fused or chemicallyconjugated (including both covalent and non-covalent conjugations) to aheterologous protein or polypeptide (or fragment thereof, preferably toa polypeptide of at least 10, at least 20, at least 30, at least 40, atleast 50, at least 60, at least 70, at least 80, at least 90 or at least100 amino acids) to generate fusion proteins. In particular, theinvention provides fusion proteins comprising an antigen-bindingfragment of an antibody described herein (e.g., a Fab fragment, Fdfragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VLdomain or a VL CDR) and a heterologous protein, polypeptide, or peptide.Methods for fusing or conjugating proteins, polypeptides, or peptides toan antibody or an antibody fragment are known in the art. See, e.g.,U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851,and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166;International Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi etal., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al.,1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad.Sci. USA 89:11337-11341.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol.287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313(each of these patents and publications are hereby incorporated byreference in its entirety). Antibodies or fragments thereof, or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. A polynucleotideencoding an antibody or fragment thereof that specifically binds to aFXIa protein may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

Moreover, the antibodies or fragments thereof can be fused to markersequences, such as a peptide to facilitate purification. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide(SEQ ID NO: 48), such as the tag provided in a pQE vector (QIAGEN, Inc.,9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many ofwhich are commercially available. As described in Gentz et al., 1989,Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine (SEQID NO: 48) provides for convenient purification of the fusion protein.Other peptide tags useful for purification include, but are not limitedto, the hemagglutinin (“HA”) tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., 1984,Cell 37:767), and the “flag” tag.

In other embodiments, antibodies of the present invention or fragmentsthereof conjugated to a diagnostic or detectable agent. Such antibodiescan be useful for monitoring or prognosing the onset, development,progression and/or severity of a disease or disorder as part of aclinical testing procedure, such as determining the efficacy of aparticular therapy. Such diagnosis and detection can accomplished bycoupling the antibody to detectable substances including, but notlimited to, various enzymes, such as, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidinlbiotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (1311, 1251, 1231, and 1211,), carbon (14C),sulfur (35S), tritium (3H), indium (1151n, 1131n, 1121n, and 111In,),technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium(103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu,159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142 Pr,105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn,75Se, 113Sn, and 117Tin; and positron emitting metals using variouspositron emission tomographies, and noradioactive paramagnetic metalions.

The present invention further encompasses uses of antibodies orfragments thereof conjugated to a therapeutic moiety. An antibody orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent ora radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety or drug moiety that modifies a given biologicalresponse. Therapeutic moieties or drug moieties are not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein, peptide, or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin; a protein such as tumor necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, an anti-angiogenicagent; or, a biological response modifier such as, for example, alymphokine.

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive metal ion, such as alph-emiters such as 213Bi ormacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, 131 In, 131LU, 131Y, 131Ho, 131Sm, to polypeptides.In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug.Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol.26(8):943-50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Methods of Producing Antibodies

Nucleic Acids Encoding the Antibodies

The invention provides substantially purified nucleic acid moleculeswhich encode polypeptides comprising segments or domains of theFXIa-binding antibody chains described above. Some of the nucleic acidsof the invention comprise the nucleotide sequence encoding the heavychain variable region shown in SEQ ID NO: 10 or 30, and/or thenucleotide sequence encoding the light chain variable region shown inSEQ ID NO: 20 or 40. In a specific embodiment, the nucleic acidmolecules are those identified in Table 1. Some other nucleic acidmolecules of the invention comprise nucleotide sequences that aresubstantially identical (e.g., at least 65, 80%, 95%, or 99%) to thenucleotide sequences of those identified in Table 1. When expressed fromappropriate expression vectors, polypeptides encoded by thesepolynucleotides are capable of exhibiting FXI and/or FXIa antigenbinding capacity.

Also provided in the invention are polynucleotides which encode at leastone CDR region and usually all three CDR regions from the heavy or lightchain of the FXIa-binding antibody set forth above. Some otherpolynucleotides encode all or substantially all of the variable regionsequence of the heavy chain and/or the light chain of the FXIa-bindingantibody set forth above. Because of the degeneracy of the code, avariety of nucleic acid sequences will encode each of the immunoglobulinamino acid sequences.

The nucleic acid molecules of the invention can encode both a variableregion and a constant region of the antibody. Some of nucleic acidsequences of the invention comprise nucleotides encoding a heavy chainsequence that is substantially identical (e.g., at least 80%, 90%, or99%) to the heavy chain sequence set forth in SEQ ID NO: 11 or 31. Someother nucleic acid sequences comprising nucleotide encoding a lightchain sequence that is substantially identical (e.g., at least 80%, 90%,or 99%) to the light chain sequence set forth in SEQ ID NO: 21 or 41.

The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequencesas described in the Examples below) encoding a FXIa-binding antibody orits binding fragment. Direct chemical synthesis of nucleic acids can beaccomplished by methods known in the art, such as the phosphotriestermethod of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiestermethod of Brown et al., Meth. Enzymol. 68:109, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859,1981; and the solid support method of U.S. Pat. No. 4,458,066.Introducing mutations to a polynucleotide sequence by PCR can beperformed as described in, e.g., PCR Technology: Principles andApplications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press,NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Inniset al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila et al.,Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods andApplications 1:17, 1991.

Also provided in the invention are expression vectors and host cells forproducing the FXI and/or FXIa-binding antibodies described above.Various expression vectors can be employed to express thepolynucleotides encoding the FXIa-binding antibody chains or bindingfragments. Both viral-based and nonviral expression vectors can be usedto produce the antibodies in a mammalian host cell. Nonviral vectors andsystems include plasmids, episomal vectors, typically with an expressioncassette for expressing a protein or RNA, and human artificialchromosomes (see, e.g., Harrington et al., Nat Genet 15:345, 1997). Forexample, nonviral vectors useful for expression of the FXIa-bindingpolynucleotides and polypeptides in mammalian (e.g., human) cellsinclude pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen,San Diego, Calif.), MPSV vectors, and numerous other vectors known inthe art for expressing other proteins. Useful viral vectors includevectors based on retroviruses, adenoviruses, adenoassociated viruses,herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barrvirus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brentet al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeldet al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding a FXIa-bindingantibody chain or fragment. In some embodiments, an inducible promoteris employed to prevent expression of inserted sequences except underinducing conditions. Inducible promoters include, e.g., arabinose, lacZ,metallothionein promoter or a heat shock promoter. Cultures oftransformed organisms can be expanded under noninducing conditionswithout biasing the population for coding sequences whose expressionproducts are better tolerated by the host cells. In addition topromoters, other regulatory elements may also be required or desired forefficient expression of a FXIa-binding antibody chain or fragment. Theseelements typically include an ATG initiation codon and adjacent ribosomebinding site or other sequences. In addition, the efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (see, e.g., Scharf et al., Results Probl. CellDiffer. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516,1987). For example, the SV40 enhancer or CMV enhancer may be used toincrease expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequenceposition to form a fusion protein with polypeptides encoded by insertedFXIa-binding antibody sequences. More often, the inserted FXI and/orFXIa-binding antibody sequences are linked to a signal sequences beforeinclusion in the vector. Vectors to be used to receive sequencesencoding FXI and/or FXIa-binding antibody light and heavy chain variabledomains sometimes also encode constant regions or parts thereof. Suchvectors allow expression of the variable regions as fusion proteins withthe constant regions thereby leading to production of intact antibodiesor fragments thereof. Typically, such constant regions are human.

The host cells for harboring and expressing the FXI and/or FXIa-bindingantibody chains can be either prokaryotic or eukaryotic. E. coli is oneprokaryotic host useful for cloning and expressing the polynucleotidesof the present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilis, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication). In addition, any number of a varietyof well-known promoters will be present, such as the lactose promotersystem, a tryptophan (trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters typicallycontrol expression, optionally with an operator sequence, and haveribosome binding site sequences and the like, for initiating andcompleting transcription and translation. Other microbes, such as yeast,can also be employed to express FXIa-binding polypeptides of theinvention. Insect cells in combination with baculovirus vectors can alsobe used.

In some preferred embodiments, mammalian host cells are used to expressand produce the FXI and/or FXIa-binding polypeptides of the presentinvention. These include any normal mortal or normal or abnormalimmortal animal or human cell. For example, a number of suitable hostcell lines capable of secreting intact immunoglobulins have beendeveloped including the CHO cell lines, various Cos cell lines, HeLacells, myeloma cell lines, and transformed B-cells. The use of mammaliantissue cell culture to express polypeptides is discussed generally in,e.g., Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y., N.Y., 1987.Expression vectors for mammalian host cells can include expressioncontrol sequences, such as an origin of replication, a promoter, and anenhancer (see, e.g., Queen, et al., Immunol. Rev. 89:49-68, 1986), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences.

These expression vectors usually contain promoters derived frommammalian genes or from mammalian viruses. Suitable promoters may beconstitutive, cell type-specific, stage-specific, and/or modulatable orregulatable. Useful promoters include, but are not limited to, themetallothionein promoter, the constitutive adenovirus major latepromoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter,the MRP polIII promoter, the constitutive MPSV promoter, thetetracycline-inducible CMV promoter (such as the human immediate-earlyCMV promoter), the constitutive CMV promoter, and promoter-enhancercombinations known in the art.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts. (See generallySambrook, et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22 (Elliot and O'Hare,Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivotransduction. For long-term, high-yield production of recombinantproteins, stable expression will often be desired. For example, celllines which stably express FXIa-binding antibody chains or bindingfragments can be prepared using expression vectors of the inventionwhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth of cells which successfully express the introducedsequences in selective media. Resistant, stably transfected cells can beproliferated using tissue culture techniques appropriate to the celltype.

Framework or Fc Engineering

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within VH and/or VL,e.g. to improve the properties of the antibody. Typically such frameworkmodifications are made to decrease the immunogenicity of the antibody.For example, one approach is to “backmutate” one or more frameworkresidues to the corresponding germline sequence. More specifically, anantibody that has undergone somatic mutation may contain frameworkresidues that differ from the germline sequence from which the antibodyis derived. Such residues can be identified by comparing the antibodyframework sequences to the germline sequences from which the antibody isderived. To return the framework region sequences to their germlineconfiguration, the somatic mutations can be “backmutated” to thegermline sequence by, for example, site-directed mutagenesis. Such“backmutated” antibodies are also intended to be encompassed by theinvention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell-epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the mutations as described in U.S. Pat. No. 6,277,375 to Wardcan be used. Alternatively, to increase the biological half-life, theantibody can be altered within the CH1 or CL region to contain a salvagereceptor binding epitope taken from two loops of a CH2 domain of an Fcregion of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al.

In a specific embodiment, an anti-FXI/FXIa antibody described herein(e.g., antibody comprising VL CDRs and VH CDRs of NOV1401) comprises ahuman IgG (e.g., IgG1) Fc region comprising two amino acidsubstitutions, D265A and P329A, to reduce the likelihood for ADCC or CDCcaused by any surface-associated FXI. These Alanine substitutions havebeen shown to reduce ADCC and CDC (see, e.g., Idosugie et al., J.Immunol. 164:4178-4184, 2000; Shields et al., J. Biol. Chem.276:6591-6604, 2001).

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for “antigen’. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lecl3 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTlll)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Methods of Engineering Altered Antibodies

As discussed above, the FXIa-binding antibodies having VH and VLsequences or full length heavy and light chain sequences shown hereincan be used to create new FXIa-binding antibodies by modifying fulllength heavy chain and/or light chain sequences, VH and/or VL sequences,or the constant region(s) attached thereto. Thus, in another aspect ofthe invention, the structural features of a FXIa-binding antibody of theinvention are used to create structurally related FXIa-bindingantibodies that retain at least one functional property of theantibodies of the invention, such as binding to human FXIa and alsoinhibiting one or more functional properties of FXIa (e.g., inhibit FXIabinding to the FXIa receptor, inhibit FXIa-dependent cellproliferation).

For example, one or more CDR regions of the antibodies of the presentinvention, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, FXIa-binding antibodies of the invention, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the VH and/or VL sequences provided herein, or one ormore CDR regions thereof. To create the engineered antibody, it is notnecessary to actually prepare (i.e., express as a protein) an antibodyhaving one or more of the VH and/or VL sequences provided herein, or oneor more CDR regions thereof. Rather, the information contained in thesequence(s) is used as the starting material to create a “secondgeneration” sequence(s) derived from the original sequence(s) and thenthe “second generation” sequence(s) is prepared and expressed as aprotein.

Accordingly, in another embodiment, the invention provides a method forpreparing a FXIa-binding antibody consisting of a heavy chain variableregion antibody sequence having a CDR1 sequence selected from the groupconsisting of SEQ ID NOs: 3 and 23, a CDR2 sequence selected from thegroup consisting of SEQ ID NOs: 4 and 24, and/or a CDR3 sequenceselected from the group consisting of SEQ ID NOs: 5 and 25; and a lightchain variable region antibody sequence having a CDR1 sequence selectedfrom the group consisting of SEQ ID NOs: 13 and 33, a CDR2 sequenceselected from the group consisting of SEQ ID NOs: 14 and 34, and/or aCDR3 sequence selected from the group consisting of SEQ ID NOs: 15 and35; altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing a FXIa-binding antibody consisting of a heavy chain variableregion antibody sequence having a CDR1 sequence selected from the groupconsisting of SEQ ID NOs: 6 and 26, a CDR2 sequence selected from thegroup consisting of SEQ ID NOs: 7 and 27, and/or a CDR3 sequenceselected from the group consisting of SEQ ID NOs: 8 and 28; and a lightchain variable region antibody sequence having a CDR1 sequence selectedfrom the group consisting of SEQ ID NOs: 16 and 36, a CDR2 sequenceselected from the group consisting of SEQ ID NOs: 17 and 37, and/or aCDR3 sequence selected from the group consisting of SEQ ID NOs: 18 and38; altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing a FXIa-binding antibody optimized for expression in amammalian cell consisting of: a full length heavy chain antibodysequence having a sequence selected from the group of SEQ ID NOs: 11 or31; and a full length light chain antibody sequence having a sequenceselected from the group of 21 or 41; altering at least one amino acidresidue within the full length heavy chain antibody sequence and/or thefull length light chain antibody sequence to create at least one alteredantibody sequence; and expressing the altered antibody sequence as aprotein. In one embodiment, the alteration of the heavy or light chainis in the framework region of the heavy or light chain.

The altered antibody sequence can also be prepared by screening antibodylibraries having fixed CDR3 sequences or minimal essential bindingdeterminants as described in US2005/0255552 and diversity on CDR1 andCDR2 sequences. The screening can be performed according to anyscreening technology appropriate for screening antibodies from antibodylibraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the FXIa-binding antibodies described herein,which functional properties include, but are not limited to,specifically binding to human, cynomolgus, rat, and/or mouse FXIa; andthe antibody inhibit FXIa-dependent cell proliferation in a F36E and/orBa/F3-FXIaR cell proliferation assay.

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an FXIa-binding antibody coding sequence and the resultingmodified FXIa-binding antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

In certain embodiments of the invention antibodies have been engineeredto remove sites of deamidation. Deamidation is known to cause structuraland functional changes in a peptide or protein. Deamindation can resultin decreased bioactivity, as well as alterations in pharmacokinetics andantigenicity of the protein pharmaceutical. (Anal Chem. 2005 Mar. 1;77(5):1432-9).

In certain embodiments of the invention the antibodies have beenengineered to increase pl and improve their drug-like properties. The plof a protein is a key determinant of the overall biophysical propertiesof a molecule. Antibodies that have low pls have been known to be lesssoluble, less stable, and prone to aggregation. Further, thepurification of antibodies with low pl is challenging and can beproblematic especially during scale-up for clinical use. Increasing thepl of the anti-FXI/FXIa antibodies, or Fabs, of the invention improvedtheir solubility, enabling the antibodies to be formulated at higherconcentrations (>100 mg/ml). Formulation of the antibodies at highconcentrations (e.g. >100 mg/ml) offers the advantage of being able toadminister higher doses of the antibodies, which in turn may enablereduced dosing frequency, a significant advantage for treatment ofchronic diseases including thrombotic and/or thromboembolic disorders.Higher pls may also increase the FcRn-mediated recycling of the IgGversion of the antibody thus enabling the drug to persist in the bodyfor a longer duration, requiring fewer injections. Finally, the overallstability of the antibodies is significantly improved due to the higherpl resulting in longer shelf-life and bioactivity in vivo. Preferably,the pl is greater than or equal to 8.2.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

Prophylactic and Therapeutic Uses

Antibodies that bind FXI and/or FXIa as described herein (e.g.,antibodies described in Table 1, such as, anti-FXI/FXIa antibodiescomprising VL CDRs and VHCDRs of NOV1401), can be used at atherapeutically useful concentration for the treatment of athromboembolic disease or disorder (e.g., thrombic stroke, atrialfibrillation, stroke prevention in atrial fibrillation (SPAF), deep veinthrombosis, venous thromboembolism, pulmonary embolism, acute coronarysyndromes (ACS), ischemic stroke, acute limb ischemia, chronicthromboembolic pulmonary hypertension, or systemic embolism) byadministering to a subject in need thereof an effective amount of theantibodies or antigen binding fragments of the invention. The presentinvention provides a method of treating thromboembolic disorder (e.g.,thrombotic disorders) by administering to a subject in need thereof aneffective amount of the antibodies of the invention. The presentinvention provides a method of treating thromboembolic disorders (e.g.,thrombic stroke, atrial fibrillation, stroke prevention in atrialfibrillation (SPAF), deep vein thrombosis, venous thromboembolism,pulmonary embolism, acute coronary syndromes (ACS), ischemic stroke,acute limb ischemia, chronic thromboembolic pulmonary hypertension, orsystemic embolism) by administering to a subject in need thereof aneffective amount of the antibodies of the invention.

The antibodies described herein (e.g., antibodies described in Table 1,such as, NOV1401 or anti-FXI/FXIa antibodies comprising VL CDRs andVHCDRs of NOV1401) can be used, inter alia, to prevent treat, prevent,and improve thromboembolic conditions or disorders, including but notlimited to thrombotic disorders, as described in greater detail herein.

The antibodies provided herein (e.g., antibodies described in Table 1,such as, anti-FXI/FXIa antibodies comprising VL CDRs and VHCDRs ofNOV1401) can also be used in combination with other agents for theprevention, treatment, or improvement of thromboembolic disorders. Forexample, statin therapies may be used in combination with the FXIaantibodies and antigen binding fragments of the invention for thetreatment of patients with thrombotic and/or thromboembolic disorders.

In a specific embodiment, provided herein is a method of treating orpreventing stroke in a patient with atrial fibrillation, comprisingadministering to the patient in need hereof an effective amount of ananti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIaantibody described in Table 1, such as, NOV1401 or anti-FXI/FXIaantibodies comprising VL CDRs and VHCDRs of NOV1401.

In a specific embodiment, provided herein is a method of managing orpreventing risks or conditions associated with atrial fibrillation (AF),such as embolic stroke and systemic embolism, in a patient with atrialfibrillation, comprising administering to the patient in need hereof aneffective amount of an anti-FXI/FXIa antibody described herein, forexample, an anti-FXI/FXIa antibody described in Table 1, such as,NOV1401 or anti-FXI/FXIa antibodies comprising VL CDRs and VHCDRs ofNOV1401.

In a specific embodiment, provided herein is a method of treating,managing or preventing conditions associated with atrial fibrillation(AF), such as embolic stroke and systemic embolism, in a patient withatrial fibrillation, comprising administering to the patient in needhereof an effective amount of an anti-FXI/FXIa antibody describedherein, for example, an anti-FXI/FXIa antibody described in Table 1,such as, NOV1401 or anti-FXI/FXIa antibodies comprising VL CDRs andVHCDRs of NOV1401. In particular embodiments, an AF patient has a highbleeding risk.

In a specific embodiment, provided herein is a method of treating,managing or preventing deep vein thrombosis or conditions associatedtherewith, in a subject (e.g., a subject with, or at risk of developing,deep vein thrombosis), comprising administering to the subject in needhereof an effective amount of an anti-FXI/FXIa antibody describedherein, for example, an anti-FXI/FXIa antibody described in Table 1,such as, NOV1401 or anti-FXI/FXIa antibodies comprising VL CDRs andVHCDRs of NOV1401.

In a specific embodiment, provided herein is a method of treating,managing or preventing venous thromboembolism (VTE) or conditionsassociated therewith, in a subject (e.g., a subject with, or at risk ofdeveloping, venous thromboembolism), comprising administering to thesubject in need hereof an effective amount of an anti-FXI/FXIa antibodydescribed herein, for example, an anti-FXI/FXIa antibody described inTable 1, such as, NOV1401 or anti-FXI/FXIa antibodies comprising VL CDRsand VHCDRs of NOV1401. In particular embodiments, subjects being treatedwith an anti-FXI/FXIa antibody provided herein have experienced 1) afirst unprovoked VTE with low risk for bleeding, 2) recurrence ofunprovoked VTE, or 3) VTE associated with thrombophilia including cancerpatients.

In a specific embodiment, provided herein is a method of treating,managing or preventing pulmonary embolism or conditions associatedtherewith, in a subject (e.g., a subject with, or at risk of developing,pulmonary embolism), comprising administering to the subject in needhereof an effective amount of an anti-FXI/FXIa antibody describedherein, for example, an anti-FXI/FXIa antibody described in Table 1,such as, NOV1401 or anti-FXI/FXIa antibodies comprising VL CDRs andVHCDRs of NOV1401.

In a specific embodiment, provided herein is a method of treating,managing or preventing acute coronary syndromes (ACS) or conditionsassociated therewith, in a subject, comprising administering to thesubject in need hereof an effective amount of an anti-FXI/FXIa antibodydescribed herein, for example, an anti-FXI/FXIa antibody described inTable 1, such as, NOV1401 or anti-FXI/FXIa antibodies comprising VL CDRsand VHCDRs of NOV1401.

In a specific embodiment, provided herein is a method of treating,managing or preventing ischemic stroke, in a subject (e.g., a subjectwith, or at risk of developing, ischemic stroke), comprisingadministering to the subject in need hereof an effective amount of ananti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIaantibody described in Table 1, such as, NOV1401 or anti-FXI/FXIaantibodies comprising VL CDRs and VHCDRs of NOV1401.

In a specific embodiment, provided herein is a method of treating,managing or preventing acute limb ischemia, in a subject, comprisingadministering to the subject in need hereof an effective amount of ananti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIaantibody described in Table 1, such as, NOV1401 or anti-FXI/FXIaantibodies comprising VL CDRs and VHCDRs of NOV1401.

In a specific embodiment, provided herein is a method of treating,managing or preventing chronic thromboembolic pulmonary hypertension, ina subject, comprising administering to the subject in need hereof aneffective amount of an anti-FXI/FXIa antibody described herein, forexample, an anti-FXI/FXIa antibody described in Table 1, such as,NOV1401 or anti-FXI/FXIa antibodies comprising VL CDRs and VHCDRs ofNOV1401.

In a specific embodiment, provided herein is a method of treating,managing or preventing systemic embolism, in a subject (e.g., a subjectwith, or at risk of developing, systemic embolism), comprisingadministering to the subject in need hereof an effective amount of ananti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIaantibody described in Table 1, such as, NOV1401 or anti-FXI/FXIaantibodies comprising VL CDRs and VHCDRs of NOV1401.

In a certain embodiment, provided herein is a method of treating,managing, or preventing thromboembolic conditions that arecatheter-related conditions (e.g., Hickman catheter in cancer patients)in which catheters become thrombosed, or extracorporeal membraneoxygenation (ECMO), in which the tubing develops clots, comprisingadministering to the subject in need hereof an effective amount of ananti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIaantibody described in Table 1, such as, NOV1401 or anti-FXI/FXIaantibodies comprising VL CDRs and VHCDRs of NOV1401.

In particular embodiments, subjects in need of treatment with ananti-FXI/FXIa antibody described herein, for example, an anti-FXI/FXIaantibody described in Table 1, such as, NOV1401 or anti-FXI/FXIaantibodies comprising VL CDRs and VHCDRs of NOV1401, may include:

-   -   Subjects with indications for chronic anticoagulation therapy        (e.g., AF, left ventricular thrombus, prior cardioembolic        stroke)    -   subjects at intermediate-to-high risk for major bleeding;    -   subjects undergoing elective or primary percutaneous coronary        intervention (PCI) with stenting which may be require to receive        dual antiplatelet therapy (aspirin and P2Y12 receptor        antagonists) to prevent stent thrombosis.

In particular embodiments, one of the following conditions can betreated or managed with an anti-FXI/FXIa antibody described herein, forexample, an anti-FXI/FXIa antibody described in Table 1, such as,NOV1401 or anti-FXI/FXIa antibodies comprising VL CDRs and VHCDRs ofNOV1401:

-   -   thromboembolism in subjects with suspected or confirmed cardiac        arrhythmia such as paroxysmal, persistent or permanent atrial        fibrillation or atrial flutter;    -   stroke prevention in atrial fibrillation (SPAF), a subpopulation        of which is AF patients undergoing percutaneous coronary        interventions (PCI);    -   acute venous thromboembolic events (VTE) treatment and extended        secondary VTE prevention in patients at high risk for bleeding;    -   cerebral and cardiovascular events in secondary prevention after        transient ischemic attack (TIA) or non-disabling stroke and        prevention of thromboembolic events in heart failure with sinus        rhythm;    -   clot formation in left atrium and thromboembolism in subjects        undergoing cardioversion for cardiac arrhythmia;    -   thrombosis before, during and after ablation procedure for        cardiac arrhythmia;    -   venous thrombosis, this includes but not exclusively, treatment        and secondary prevention of deep or superficial veins thrombosis        in the lower members or upper member, thrombosis in the        abdominal and thoracic veins, sinus thrombosis and thrombosis of        jugular veins;    -   thrombosis on any artificial surface in the veins like catheter        or pacemaker wires;    -   pulmonary embolism in patients with or without venous        thrombosis;    -   Chronic Thromboembolic Pulmonary Hypertension (CTEPH);    -   arterial thrombosis on ruptured atherosclerotic plaque,        thrombosis on intra-arterial prosthesis or catheter and        thrombosis in apparently normal arteries, this includes but not        exclusively acute coronary syndromes, ST elevation myocardial        infarction, non ST elevation myocardial infarction, unstable        angina, stent thrombosis, thrombosis of any artificial surface        in the arterial system and thrombosis of pulmonary arteries in        subjects with or without pulmonary hypertension;    -   thrombosis and thromboembolism in patients undergoing        percutaneous coronary interventions (PCI);    -   cardioembolic and cryptogenic strokes;    -   thrombosis in patients with invasive and non-invasive cancer        malignancies;    -   thrombosis over an indwelling catheter;    -   thrombosis and thromboembolism in severely ill patients;    -   cardiac thrombosis and thromboembolism, this includes but not        exclusively cardiac thrombosis after myocardial infarction,        cardiac thrombosis related to condition such as cardiac        aneurysm, myocardial fibrosis, cardiac enlargement and        insufficiency, myocarditis and artificial surface in the heart;    -   thromboembolism in patients with valvular heart disease with or        without atrial fibrillation;    -   thromboembolism over valvular mechanic or biologic prostheses;    -   injuries or trauma in patients who had native or artificial        cardiac patches, arterial or venous conduit tubes after heart        repair of simple or complex cardiac malformations;    -   venous thrombosis and thromboembolism after knee replacement        surgery, hip replacement surgery, and orthopedic surgery,        thoracic or abdominal surgery;    -   arterial or venous thrombosis after neurosurgery including        intracranial and spinal cord interventions;    -   congenital or acquired thrombophilia including but not        exclusively factor V Leiden, prothrombin mutation, antithrombin        III, protein C and protein S deficiencies, factor XIII mutation,        familial dysfibrinogenemia, congenital deficiency of        plasminogen, increased levels of factor XI, sickle cell disease,        antiphospholipid syndrome, autoimmune disease, chronic bowel        disease, nephrotic syndrome, hemolytic uremia,        myeloproliferative disease, disseminated intra vascular        coagulation, paroxysmal nocturnal hemoglobinuria and heparin        induced thrombopenia;    -   thrombosis and thromboembolism in chronic kidney disease; and    -   thrombosis and thromboembolism in patients undergoing        hemodialysis and extra-corporal membrane oxygenation.

In a specific aspect, provided herein are methods of managing bleedingin a patient being treated or administered an anti-FXI/FXIa antibodyprovided herein (e.g., an antibody described in Table 1, such as, ananti-FXI/FXIa antibody comprising VL CDRs and VHCDRs of NOV1401), forexample, bleeding associated with trauma, surgery menstruation orpost-delivery, said method comprises reversing of the anticoagulanteffect. FXI deficiency is rarely associated with spontaneous bleedingmanifestations; in specific aspects, bleeding is most typicallyassociated with trauma, surgery, menstruation or post-delivery.Prolonged bleeding may occur after a major trauma or after surgeryinvolving organs with high fibrinolytic area such as buccal, nasal,genital or urinary mucosa. Tooth extraction, tonsillectomy and ablationof the uterus or prostate are examples of surgeries that entail a highrisk of bleeding. People with the disorder also have a strong tendencyto develop epistaxis and ecchymoses, and more rarely, bleeding into theurine or intestines. Spontaneous muscle or joint and intracranialbleeding frequency is not increased in patients with FXI deficiency.Venous puncture is not usually associated with an extended bleeding.Other genetic mutations associated with FXI deficiency may contribute tothe heterogeneous and unpredictable bleeding tendency in patients withsevere FXI deficiency. Concomitant use of antiplatelets, otheranticoagulants and fibrinolytic agents can increase the risk ofbleeding.

In particular embodiments, provided herein is a method of managingbleeding in a patient being treated with an anti-FXI/FXIa antibodyprovided herein (e.g., an antibody described in Table 1, such as, ananti-FXI/FXIa antibody comprising VL CDRs and VHCDRs of NOV1401), saidmethod comprises temporarily reversing of the anticoagulant effect for asufficient time to manage the bleeding. In specific embodiments, thestep of reversing of the anticoagulant effect comprises (i) fluidreplacement using colloids, crystalloids, human plasma or plasmaproteins such as albumin; or (ii) transfusion with packed red blood orwhole blood. In a particular embodiment, therapeutic agents for reversalof the effect of anticoagulants, for example, in cases of severeemergency, include, but are not limited to, prohemostasis bloodcomponents such as fresh frozen plasma (FFP), prothrombin complexconcentrates (PCC) and activated PCC [(APCC); e.g. factor VIII inhibitorbypass activity (FEIBA)] and recombinant activated factor VII (rFVIIa).In one embodiment, a regimen comprising administration of rFVIIa at adose of 30 μg/kg followed by administration of rFVIIa at a dose of 15-30μg/kg every 2-4 hours for 24-48 hours in addition to tranexamic acid 1 gevery 6 hours for 5 to 7 days may have potential to recover hemostasisand stop bleeding in subjects treated with an anti-FXI/FXIa antibodyprovided herein (e.g., NOV1401 or an antibody comprising VL CDRs and VHCDRs of NOV1401) who are undergoing major surgery and in patients withan active non-accessible bleeding site. For instance, Riddell et alreported experience in 4 patients with severe FXI deficiency withoutinhibitor undergoing surgery (Riddell et al., 2011, Thromb. Haemost.;106: 521-527); patients were administered rFVIIa 30 μg/kg and tranexamicacid 1 g i.v. at induction of anesthesia. Subsequent bolus doses ofrFVIIa 15-30 μg/kg were administered at 2 to 4 hourly intervals asguided by rotational thromboelastometry (ROTEM) results. Patients weretreated with rFVIIa at above mentioned doses for 24-48 hours. Tranexamicacid 1 g every six-hourly was continued for five days. In this smallseries, rFVIIa at doses as low as 15-30 μg/kg in combination withtranexamic acid was safe and effective in correcting the hemostaticdefect in severe FXI deficiency in this study. In another studycomprising 4 patients with severe FXI deficiency with inhibitor(autologous neutralizing FXI antibodies usually acquired aftertransfusion or administration of blood products to patients with severeFXI deficiency) who experienced 5 surgeries, the authors (Livnat et al.,2009, Thromb. Haemost.; 102: 487-492) applied the following protocol: 1g of tranexamic acid orally two hours before surgery, then patientsreceived immediately prior to the interventions another 1 g tranexamicacid i.v. Recombinant FVIIa at doses ranging from 15 to 30 μg/kg wasinfused at the completion of surgery. Subsequently, oral tranexamic acid1 g was given every 6 hour for at least 7 days. Fibrin glue was sprayedat the bed of the extirpated gallbladder in one patient. This protocolsecured normal hemostasis in patients with severe FXI deficiency withinhibitor.

In one aspect, fibrin glue can be used to restore local hemostasisduring dental surgery in patients with FXI deficiency (Bolton-Maggs(2000) Haemophilia; 6 (S1):100-9). In a certain embodiment with respectto methods to manage bleeding in patients being treated with ananti-FXI/FXIa antibody provided herein (e.g., NOV1401), a regimenconsisting of tranexamic acid 1 g every 6 hours for 5 to 7 daysassociated with the use of fibrin glue could be used to establish localhemostasis in subjects undergoing minor surgery and in subjects withaccessible bleeding site, including oral and nasal bleeding events.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising theFXIa-binding antibodies (intact or binding fragments) formulatedtogether with a pharmaceutically acceptable carrier. The compositionscan additionally contain one or more other therapeutic agents that aresuitable for treating or preventing, for example, thromboembolicdisorders (e.g., thrombotic disorders). Pharmaceutically acceptablecarriers enhance or stabilize the composition, or can be used tofacilitate preparation of the composition. Pharmaceutically acceptablecarriers include solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible.

A pharmaceutical composition of the present invention can beadministered by a variety of methods known in the art. The route and/ormode of administration vary depending upon the desired results. It ispreferred that administration be intravenous (i.v.), intramuscular(i.m.), intraperitoneal (i.p.), or subcutaneous (s.c.), or administeredproximal to the site of the target. The pharmaceutically acceptablecarrier should be suitable for intravenous, intramuscular, subcutaneous,parenteral, spinal or epidermal administration (e.g., by injection orinfusion). Depending on the route of administration, the activecompound, i.e., antibody, bispecific and multispecific molecule, may becoated in a material to protect the compound from the action of acidsand other natural conditions that may inactivate the compound.

In particular aspects, anti-FXI/FXIa antibodies described herein (e.g.,antibodies described in Table 1, such as NOV1401 or antibodiescomprising LCDRs and HCDRs of NOV1401) are formulated at approximately75 mg/l mL to approximately 200 mg/l mL concentration, in liquid vialsfor subcutaneous injections. In particular embodiments, thepharmaceutical composition comprises a pharmaceutical carrier orexcipient, for example, sucrose, and polysorbate 20. In particularembodiments, the pharmaceutical composition comprises L-histidine and/orhistidine HCl monohydrate. In certain embodiments, the pharmaceuticalcomposition has a pH of approximately 4 to 7, or 5 to 6.

In particular aspects, anti-FXI/FXIa antibodies described herein (e.g.,antibodies described in Table 1, such as NOV1401 or antibodiescomprising LCDRs and HCDRs of NOV1401) are formulated at 150 mg/l mLconcentration, in liquid vials for subcutaneous injections. In oneembodiment, the 150 mg/mL liquid formulation contains 150 mganti-FXI/FXIa antibody, L-histidine, histidine HCl monohydrate, sucrose,and polysorbate 20, with a pH=5.5±0.5. The composition should be sterileand fluid. Proper fluidity can be maintained, for example, by use ofcoating such as lecithin, by maintenance of required particle size inthe case of dispersion and by use of surfactants. In many cases, it ispreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol or sorbitol, and sodium chloride in the composition.Long-term absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Pharmaceutical compositions of the invention can be prepared inaccordance with methods well known and routinely practiced in the art.See, e.g., Remington: The Science and Practice of Pharmacy, MackPublishing Co., 20th ed., 2000; and Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978. Pharmaceutical compositions are preferably manufacturedunder GMP conditions. Typically, a therapeutically effective dose orefficacious dose of the FXIa-binding antibody is employed in thepharmaceutical compositions of the invention. The FXIa-bindingantibodies are formulated into pharmaceutically acceptable dosage formsby conventional methods known to those of skill in the art. Dosageregimens are adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level depends upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, thereof, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors.

A physician can start doses of the antibodies of the invention employedin the pharmaceutical composition at levels lower than that required toachieve the desired therapeutic effect and gradually increase the dosageuntil the desired effect is achieved. In general, effective doses of thecompositions of the present invention, for the treatment of a thromboticand/or thromboembolic disorders described herein vary depending uponmany different factors, including means of administration, target site,physiological state of the patient, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. For systemicadministration with an antibody, the dosage ranges from about 0.01 to 15mg/kg of the host body weight. For administration (e.g., subcutaneousadministration) with an antibody, the dosage may range from 0.1 mg to 5mg or from 1 mg to 600 mg. For example, an anti-FXI/FXIa antibodydescribed herein can be administered at a dose of 0.1 mg/kg, 0.2 mg/kg,0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg,1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg,2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg, 3.4 mg/kg, 3.5mg/kg, 3.6 mg/kg, 3.7 mg/kg, 3.8 mg/kg, 3.9 mg/kg, 4.0 mg/kg, 4.1 mg/kg,4.2 mg/kg, 4.3 mg/kg, 4.4 mg/kg, 4.5 mg/kg, 4.6 mg/kg, 4.7 mg/kg, 4.8mg/kg, 4.9 mg/kg, or 5.0 mg/kg. An exemplary treatment regime entailssystemic administration once per every two weeks or once a month or onceevery 3 to 6 months. An exemplary treatment regime entails systemicadministration once per week, once per every two weeks, once per everythree weeks, once a month, or once every 3 to 6 months, or as needed(PRN).

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby i.v. or s.c., at a dose of 3 mg/kg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby i.v. or s.c., at a dose of 10 mg/kg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby i.v. or s.c., at a dose of 30 mg/kg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby i.v. or s.c., at a dose of 50 mg/kg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby i.v. or s.c., at a dose of 100 mg/kg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby i.v. or s.c. route, at a dose in the range of 5 mg to 600 mg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby i.v. or s.c. route, at a dose of approximately 5 mg, 10 mg, 15 mg, 20mg, 30 mg, 40 mg, 50 mg, 60 mg, 90 mg, 100 mg, 120 mg, 150 mg, 180 mg,200 mg, 210 mg, 240 mg, 250 mg, 270 mg, 300 mg, 330 mg, 350 mg, 360 mg,390 mg, 400 mg, 420 mg, 450 mg, 480 mg, 500 mg, 510 mg, 540 mg, 550 mg,570 mg, or 600 mg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby s.c. route, at a dose of 5 mg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby s.c. route, at a dose of 15 mg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby s.c. route, at a dose of 50 mg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby s.c. route, at a dose of 150 mg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby s.c. route, at a dose of 300 mg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby s.c. route, at a dose of 600 mg.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby i.v. or s.c. route, at a dose sufficient to achieve a mean durationof aPTT prolongation of 2-fold or greater for a period no longer than 30days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42days, 43 days, 44 days, 45 days, or 50 days.

In a certain embodiment, an anti-FXI/FXIa antibody described herein(e.g., an antibody described in Table 1, such as NOV1401 or an antibodycomprising VL CDRs and VH CDRs of NOV1401) is administered, for exampleby i.v. or s.c. route, at a dose sufficient to achieve a mean durationof aPTT prolongation of 2-fold or greater for a period no longer than 42days.

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, biweekly, monthly or yearly.Intervals can also be irregular as indicated by measuring blood levelsof FXI- and/or FXIa-binding antibody in the patient. In additionalternative dosing intervals can be determined by a physician andadministered monthly or as necessary to be efficacious. In some methodsof systemic administration, dosage is adjusted to achieve a plasmaantibody concentration of 1-1000 μg/mL or 1-1200 μg/mL, and in somemethods 25-500 μg/mL. Alternatively, antibody can be administered as asustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody and its target in the patient. In general,human and humanized antibodies show longer half-life, in humans, thanthat of chimeric antibodies and nonhuman antibodies. The dosage andfrequency of administration can vary depending on whether the treatmentis prophylactic or therapeutic. In prophylactic applications, arelatively low dosage is administered at relatively infrequent intervalsover a long period of time. Some patients continue to receive treatmentfor the rest of their lives. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregimen.

EXAMPLES

The following examples are provided to further illustrate the inventionbut not to limit its scope. Other variants of the invention will bereadily apparent to one of ordinary skill in the art and are encompassedby the appended claims.

Example 1 Human Fab Phage Library Panning

For the selection of antibodies recognizing human Factor XI, multiplepanning strategies were utilized. Therapeutic antibodies againstdifferent variants of human Factor XI and rabbit Factor XIa catalyticdomain protein were generated by the selection of clones that bound toFactor XI using as a source of antibody a commercially available phagedisplay library, the Morphosys HuCAL PLATINUM® library. The phagemidlibrary is based on the HuCAL® concept (Knappik et al., 2000, J Mol Biol296: 57-86) and employs the CysDisplay™ technology for displaying theFab on the phage surface (WO01/05950). For the isolation of anti-FactorXI antibodies liquid phase panning strategies were employed.

Cross-Reactivity Analysis

Purified Fabs were tested in ELISA for binding to the different variantsof human Factor XI (Factor XI, Factor XIa & Factor XIa catalytic domain)and Rabbit Factor XIa catalytic domain biotinylated proteins. For thispurpose Maxisorp™ (Nunc) 384 well plates were coated with 10 ug/ml ofNeutrAvidin in PBS overnight at 4° C. Antigens were captured on theNeutrAvidin via the biotin at room temperature (RT) for 30 minutes.Binding of Fabs at different concentrations was detected by F(ab)₂specific goat anti-human IgG conjugated to alkaline phosphatase (diluted1:5000) using Attophos fluorescence substrate (Roche, catalog#11681982001). Fluorescence emission at 535 nm was recorded withexcitation at 430 nm.

Conversion to IgG and IgG Expression

In order to express full length IgG in CAP-T cells, variable domainfragments of heavy (VH) and light chains (VL) were subcloned from Fabexpression vectors into appropriate pMorph®_hlg vectors for human IgG1.Two amino acid substitutions (D265A and P329A) were introduced in the Fcportion to reduce the likelihood for ADCC or CDC caused by anysurface-associated FXI. These Alanine substitutions have been shown toreduce ADCC and CDC (see, e.g., Idosugie et al., J. Immunol.164:4178-4184, 2000; Shields et al., J. Biol. Chem. 276:6591-6604,2001). The cell culture supernatant was harvested 7 days posttransfection. After sterile filtration, the solution was subjected toProtein A affinity chromatography using a liquid handling station.Samples were eluted in a 50 nM Citrate, 140 nM NaOH and pH neutralizedwith 1M Tris buffer and sterile filtered (0.2 μm pore size). Proteinconcentrations were determined by UV-spectrophotometry at 280 nm andpurity of IgGs was analyzed under denaturing, reducing conditions inSDS-PAGE.

Example 2 Binding Data

Surface Plasmon Resonance (SPR) Analysis for the FXI Catalytic Domain.

The SPR measurements were performed on a BIACORE™ T200 surface plasmonresonance based optical biosensor (BIACORE™, GE Healthcare, Uppsala).Series S sensor chips (CM5), immobilization kits and regeneration bufferwere purchased from GE Healthcare (Uppsala). Two different assay setupswere performed depending on the ligand format, IgG or Fab. First, thesurface was activated by N-hydroxysuccinimide (NHS) andN-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC). TheNOV1401-Fab was covalently attached to the activated dextran matrix on aCM5 chip by the standard amine-coupling method (GE Healthcare, Uppsala).For the NOV1401-IgG a capture assay was performed and a goat anti-humanIgG-Fc antibody (JIR) was immobilized on the chip at 14000 RUs.Remaining active surface groups were inactivated with Ethanolamine (EA).A reference cell without immobilized ligand was prepared and the systemequilibrated with 1×HBS-EP+buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA,0.05% P20, pH 7.4, Teknova H8022).

All binding experiments were performed at 25° C. at a flow rate of 50μL/min using HBS-EP+buffer. For the capture assay NOV1401-IgG wascaptured until reaching an RU level of 80. For kinetic studies adilution series of the FXI catalytic domain with concentrations rangingfrom 0-200 nM in HBS-EP+buffer was used. Association time was 120 s andthe dissociation time 180 s. The surface was regenerated with a singleinjection of 10 mM Glycine pH 1.5 (contact time 60 s, stabilization time120 s). Data processing as well as k_(on), k_(off), and K_(D)determination were accomplished with the T200 BiaEvaluation softwareversion 1.0. Double referencing (subtraction of reference and blankinjection) was applied to correct for bulk effects and other systematicartefacts. Sensograms were fitted by applying a 1:1 binding model(R_(max) set at global).

Solution Equilibrium Titration (SET) for FXI and FXIa

22 serial 1.6n dilutions of the antigens were prepared in sample buffer(PBS pH 7.4 containing 0.5% BSA and 0.02% Tween 20) and a constantconcentration of NOV1401-Fab (200 pM for huFXI and 500 pM for huFXIa) orNOV1401-Antibody (10 pM for huFXI and huFXIa) was added to each antigenconcentration. Starting concentrations for the antigen dilution serieswere 100 nM for huFXIa and 20 nM huFXI (Fab assay) or 1 nM (IgG assay).60 μl/well of each dilution mix was distributed in duplicates to a384-well polypropylene MTP. Sample buffer served as negative control anda sample containing no antigen as positive control (Bmax). The plate wassealed and incubated overnight at RT on a shaker. A standard 384-wellMSD array MTP was coated with 30 μl/well of 0.1 μg/ml huFXIa (for huFXIaand huFXI) diluted in PBS, sealed and incubated overnight at 4° C.

After incubation and three times washing with TBST (TBS containing 0.05%Tween 20) the antigen-coated MSD plate was blocked with 50 μl/wellblocking buffer (PBS containing 5% BSA) and incubated for 1 h at RT on ashaker. The wash step was repeated and 30 μl/well of theFab-/IgG-antigen preparation was transferred from the polypropylene MTPto the antigen coated MSD plate and incubated for 20 min at RT on ashaker. After an additional wash step, 30 μl of 0.5 μg/ml ECL-labeledgoat anti-human-IgG/Fab detection antibody (MSD) diluted in samplebuffer was added to each well and incubated for 30 min at RT withshaking. After washing the plate again three times, 35 μl of read buffer(MSD) was added to each well. Electrochemiluminescence (ECL) signalswere generated and detected with the MSD Sector Imager 6000.

Average ECL-signals were calculated from duplicate measurements withineach assay. Data were baseline adjusted by subtracting the lowest valuefrom all data points and plotted against the corresponding antigenconcentration. K_(D) values were determined by fitting the plot with thefollowing 1:1 (for Fab) or 1:2 (for IgG) fit model (according to Piehleret al., 1997).

Results

The results are summarized in Tables 3 and 4. For both NOV1401 formats,Fab and IgG, K_(D) values of approximately 20 nM were obtained for theFXI catalytic domain as determined by BIACORE™. Affinities of the Fab toboth the activated and zymogen FXI were in the pM range and were 66 and300 times higher than the affinity to the catalytic domain,respectively. Based on their high affinities these interactions weremeasured by SET assays. The NOV1401-Fab exhibited a five-fold higheraffinity to the zymogen FXI (62 pM) than to the activated FXI (305 pM).Affinities of the NOV1401-IgG to both the dimeric zymogen and activatedFXI are marked as apparent K_(D) values since the interaction mightinfluenced by avidity effects.

To confirm that NOV2401 also binds to cynomolgus monkey FXI, SETexperiments were performed with activated cynomolgus monkey FXI andcynomolgus monkey FXI zymogen resulting in apparent K_(D) values of12.5±6.6 pM (N=2) and 5.0±0.7 pM (N=2), respectively. Hence, theaffinities of NOV1401 to cynomolgus monkey FXI proteins (active form andzymogen) are comparable to those for binding to human FXI (Table 3).

TABLE 2 K_(D) values and kinetic binding parameters of NOV1401-Fab/IgGfor human FXI catalytic domain as determined by BIACORE ™. Catalyticdomain k_(a) (1/Ms) k_(d) (1/s) K_(D) (nM) n NOV1401-Fab 3.2 ± 0.5E+46.1 ± 1.8E−4 19 ± 6 3 NOV1401-IgG 4.2 ± 1.6E+4 8.8 ± 2.2E−4 21 ± 3 2

TABLE 3 K_(D) values of NOV1401-Fab/IgG for human FXI activated andzymogen determined by the in solution equilibrium titration (SET). K_(D)[pM] n NOV1401-Fab Human FXI activated 305 ± 8   3 Human FXI zymogen 62± 18  3 NOV1401-IgG Human FXI activated 4.7 ± 2.1* 3 Human FXI zymogen1.3 ± 0.3* 3 *Only apparent K_(D) values reported, since the interactionmight be influenced by avidity effects. Reference: Piehler et al.Assessment of affinity constants by rapid solid phase detection ofequilibrium binding in a flow system, J. Immunol. Meth. 1997. 189-206

Example 3 Biochemical Assay: Inhibition of FXIa in Activity Assay UsingFluorescent Peptide as Substrate

The activity of human FXIa (Kordia Life Science NL, catalogue numberHFXIa 1111a) is determined by monitoring the cleavage of a fluorescentlylabelled peptide with the sequence D-Leu-Pro-Arg*Rh110-D-Pro (productnumber BS-2494; Biosyntan GmbH, Berlin, Germany). In the substratesequence written above, * indicates the scissile bond, D-Leu: D-leucine,Pro: proline, Arg: arginine, Rh110: rhodamine 110, D-Pro: D-proline).FXIa mediated cleavage of the scissile bond of the peptide substrateleads to an increase of fluorescence intensity of the rhodamine 110 whenusing excitation and emission wavelengths of 485 nm and 535 nm,respectively. Fluorescence intensity is continuously measured using themicrotiter plate reader Safire2 (TECAN, Maennedorf, Switzerland) at roomtemperature (RT). The assay buffer contains 50 mM HEPES at pH 7.4, 125mM NaCl, 5 mM CaCl₂ and 0.05% (w/v) CHAPS. In the final activity assay,human FXIa and the substrate BS-2494 have assay concentrations of 0.1 nMand 0.5 μM, respectively. Under these conditions, the increase offluorescence intensity over time is linear for at least 60 minutes.

For testing the inhibitory activity of antibodies, serial dilutions ofantibodies are prepared in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mMNa₂HPO₄, 1.8 mM KH₂PO₄) containing 0.05% (w/v) CHAPS. Two μL of antibodysolution are pre-incubated with 10 μL FXIa solution (in assay buffer)for 60 minutes at RT. After the pre-incubation step, 10 μL of substrateBS-2494 (diluted in assay buffer) is added, and the enzymatic reactionis allowed to proceed for 60 minutes, after which the fluorescenceintensity is measured. The fluorescence intensity values are convertedinto percent inhibition by using control reactions (signal ofuninhibited reactions is equivalent to 0% inhibition, and a reactioncontaining no enzyme is equivalent to 100% inhibition) and the followingformula for transferring values:

y=100%−[FI(x)−FI(min)]/[FI(max)−FI(min)],

where y is the %-inhibition at the antibody concentration x, FI(x) isthe fluorescence intensity measured at antibody concentration x, FI(min)is the fluorescence intensity measured in the control reaction inabsence of antibody and FI(max) is the fluorescence intensity measuredin the uninhibited control reaction. Data are analyzed using the programOrigin 7.5SR6 (OriginLab Corporation, USA). IC50 values from averageddata are calculated using the logistics function:

y=A2+(A1−A2)/(1+(x/IC50)̂p),

where y is the %-inhibition at the antibody concentration x, A1 is thelowest inhibition value, and A2 the maximum inhibition value. Theexponent, p, is the Hill coefficient.

FIG. 6A shows a representative compound response curve of antibodyNOV1401 inhibiting the enzymatic activity of full length human FXIa. Theresults show that NOV1401 inhibits the enzymatic activity of human fulllength FXIa in a concentration dependent manner (FIG. 6A). Fitting withthe logistic fit model leads to an IC₅₀ value of approximately 160 pM.

Example 4 Anticoagulant Activity of Anti-FXIa Abs

The antithrombotic activity of the antibodies NOV1401 and NOV1090 weretested by using the activated partial thromboplastin time (aPTT) assayand the thrombin generation assay (TGA).

Aptt Assay:

Lyophilized normal human plasma ‘Coagulation Control N’ (reference no5020050) was purchased from Technoclone GmbH (Vienna, Austria). It waspooled from citrated plasma of selected healthy donors. The clottingtime obtained with this normal plasma reflects normal concentrations ofthe coagulation factors involved in clotting. The lyophilized plasma wasstored at 4° C. Prior to its use, the plasma was re-suspended in 1 mL ofdistilled water by carefully rotating the vial and then keeping it for10 minutes at RT.

The intrinsic pathway triggering reagent ‘aPTT-s’ (reference no TE0350)was purchased from SYCOmed (Lemgo, Germany) and contains phospholipidand silicate (colloidal) in a buffered solution (sodium chloride,polyethylene glycol 20000; sucrose, sodium azide). The solution wasstored at 4° C.

Calcium Chloride (reference no C1016-500G; Sigma-Aldrich Chemie GmbH,Steinheim, Germany) was prepared in bi-distillated water at a stockconcentration of 25 mM.

UltraPure Tris/HCl buffer at pH 7.5 (reference no 15567-027; LifeTechnologies Corporation, NY, USA) and Phosphate Buffered Saline (PBS,reference no P4417-100TAB; Sigma-Aldrich Chemie GmbH, Steinheim,Germany) were compound dilution.

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate(CHAPS, reference no C3023-25G) and anhydrous Dimethyl sulfoxide (DMSO,reference no 276855-100ML) were purchased from Sigma-Aldrich Chemie GmbH(Steinheim, Germany).

The measurements of the clotting time were performed in an Amelung ballcoagulometer model KC4A (purchased through SYCOmed, Lemgo, Germany),which is a semi-automated mechanical clot detection system. The systemutilizes a special cuvette (reference no A14000; SYCOmed) in which astainless steel ball (reference no A15000; SYCOmed) was placed.

The sample is added to the cuvette. After an appropriate incubationperiod, the cuvette is placed into the measuring well of the ballcoagulometer. The measuring well rotates slowly causing the cuvette torotate along its longitudinal axis. Because the cuvette is positioned ata slight angle, gravity and inertia always position the ball at thelowest point of the cuvette. Exactly opposite the ball-position is amagnetic sensor. With the addition of the trigger reagent, a timer isstarted. As coagulation takes place fibrin strands form in the reactionmixture. The fibrin strands pull the ball away from its inertia positionthat triggers an impulse in the magnetic sensor. This impulseelectronically stops the timer. The pipetting scheme was as follows(Table 4a):

TABLE 4a aPIT assay Assay step Solution Volume [μL] 1 compound dilutionor diluent 50 2 human blood plasma 50 3 aPTT-s reagent 50 4 Incubate 3minutes at 37° C. under rotation 5 25 mM Calcium Chloride 50 6Immediately start the timer 7 The timer stops when the clot is formed

The samples were measured in duplicates at a temperature of 37° C. inthe Amelung ball coagulometer.

FIG. 6B shows a representative compound response curve of antibodyNOV1401, leading to the concentration dependent prolongation of aPTTclotting times. The results suggest that NOV1401 leads to theprolongation of aPTT clotting times of human plasma in a concentrationdependent manner. The aPTT clotting time is doubled compared to baselineat a NOV1401 concentration of approximately 14 nM. The IC₅₀ value wascalculated to be approximately 13 nM.

Thrombin Generation Assay (TGA):

For the TGA lyophilized normal human plasma (Coagulation control N) waspurchased from Technoclone GmbH, (reference number 5020040, Lot#1P37600)and reconstituted in distilled water in a the volume suggested by themanufacturer.

The substrate solution was prepared using the fluorogenic substrateZ-Gly-Gly-Arg-AMC from Technoclone GmbH (reference number 5006230,Lot#8F41600). Aliquots of the lyophilized substrate were kept at 4° C.The substrate was dissolved freshly in the volume of distilled waterindicated on the vial 20 minutes prior its use in the assay. Thereconstituted substrate solution contains the fluorogenic peptide at aconcentration of 1 mM and CaCl₂ at a concentration of 15 mM.

Two different reagents ‘TGA RD’ (reference no 500622) and ‘TGA RC low’(reference no 5006213) for triggering the intrinsic and the extrinsicpathway, respectively, were purchased from Technoclone GmbH (Vienna,Austria). The trigger reagent ‘platelet poor plasma (PPP)-reagent low’was purchased from Thrombinoscope (TS31.00, Lot# PPL1409/01) andreconstituted in distilled water as indicated on the vial. PPP-reagentlow′ contains a mixture of phospholipids and tissue factor at very lowconcentration. The reagent was 8-fold diluted in 80 mM Tris/HCl atpH7.4, 0.05% (w/v) CHAPS immediately before use.

The samples were aliquoted and measured in 96 well black/clear bottomplates purchased from Costar (product no 3603). For automation transfersamples were placed in V-bottom 96 well plate (Costar, 3894) andtransferred using a CyBio automation system (Analytik Jena US, Woburn,Mass., USA).

The reconstituted human blood plasma, trigger reagent PPP-reagent low′and substrate were pre-warmed for 10 minutes in a water bath at 37° C.Serial 1:3 antibody dilutions in PBS were prepared in a 96 well platestarting with a NOV1401 concentration of 5 μM (5× the highest finalconcentration of 1 μM) for a total of 8 dilutions. 222 μl of triggerreagent was mixed with 1108 μl of substrate solution to generate the10+50 trigger reagent substrate mix. 80 μl per well was added into aV-bottom 96 well plate for later transfer using an automation system.The plate was kept at 37° C. The reagents were added according to thescheme given in Table 4b.

TABLE 4b Assay step Solution Volume [ul] 1 Antibody solutions (8dilutions) 20 2 Plasma stock solution 20 5 minutes incubation at 37° C.in a thermomixer at 300 rpm. 3 Trigger reagent/substrate mixture 10 + 50

Trigger/substrate mixtures were transferred using automation. Afteradding the mixtures, excitation and emission at 360 nm at 460 nm,respectively, were recorded immediately using a Synergy Neo instrument(BioTek Instrument Inc., Winooski, Vt., USA). The samples were measuredin duplicates at a temperature of 37° C. in the plate reader for 90minutes at intervals of 55 seconds.

To generate peak thrombin concentration values data were processed usingthe TGA evaluation software file provided by Technoclone. To generateplots for peak thrombin concentration vs antibody concentration datawere fit using GraphPad software. These data were fit to a non-linearregression model in the GraphPad Prism5 software (GraphPad SoftwareInc., La Jolla, Calif., USA). The IC₅₀ value was determined using thebuilt-in four-parameter dose-response curve equation (variable slope):y=Bottom+(Top−Bottom)/(1+10̂((LogIC50−x)*HillSlope)) where y is themaximal concentration of thrombin formed at the inhibitor concentration,x, and top and bottom represent the concentration of thrombin withoutinhibitor and at the highest concentration of inhibitor, respectively.

FIG. 6C shows a representative compound response curve of antibodyNOV1401, displaying the concentration-dependent inhibition of thrombingeneration in the TGA. An IC₅₀ value of 24 nM and a residual thrombinconcentration of 159 nM (dotted line) were calculated for this compoundresponse curve.

Example 5 Protein Expression, Complex Formation, Crystallization andStructure Determination of NOV1401 (Fab)—FXI (Catalytic Domain)

The structure of the Fab portion of the antibody NOV1401, obtained bypapain cleavage in complex with the factor XI catalytic domain wasobtained by cocrystallization at a resolution of 2.04 Å.

Protein Expression:

The expression construct for the FXI catalytic domain consisted of aminoacid residues 388-625 (Swissprot P03951) with the unpaired cysteine,C500, mutated to cysteine, with an N-terminal extension comprised of theamino acids MGSS (SEQ ID NO:49), an octa-histidine tag (SEQ ID NO: 50),a PreScission™ cleavage site followed by an enterokinase cleavage site.The construct was assembled by gene synthesis, cloned into the pET24aexpression vector and expressed as inclusion bodies in E. coli strainBL21 (DE3) grown in LB-medium. The inclusion bodies were solubilized in50 mM Tris/HCl pH 8.0, 6.0 M Guanidinium Chloride, 50 mM DTT for 2 hr.,fully denaturing the recombinant protein. A large excess of refoldingbuffer (0.5 M Tris pH 8.0, 0.9 M Arginine HCl, 5 mM GSH, 0.5 mM GSSG, 1mM EDTA was added rapidly to the IB solution to give a final proteinconcentration of 50 ug/ml and incubated at 4° C. for 5 days. Refoldingand disulfide bridge formation were accomplished by dialysis with BufferA (50 mM Tris pH 8.0) for three days.

The refolded protein was loaded onto an anion-exchange chromatographycolumn containing Q-Sepharose® FF (GE Healthcare) equilibrated andwashed with Buffer A. The unbound recombinant protein was collected fromthe flowthrough and wash fractions. The pH was adjusted to 7.4 bydialysis with 50 mM Tris pH 7.4 prior to removal of the N-terminal tagsequence using enterokinase (enterokinase: recombinant protein ratio1:100, 2.5 h incubation time). The cleavage reaction was stopped byloading the sample onto a Benzamidine affinity column containingBenzamidine Sepharose 4 FF, high sub (GE Healthcare) equilibrated andwashed with Buffer B (50 mM Tris pH 7.4, 0.5 M NaCl) and eluted withBuffer C (Buffer B containing 50 mM Benzamidine). The active FXIcatalytic domain was loaded onto an XK 26/600 Superdex 75 size exclusioncolumn (GE Healthcare) equilibrated with 20 mM Na-Acetate pH 5.3, 75 mMNaCl. The final protein concentration was 1.07 mg/ml.

The Fab portion of NOV1401 was obtained by papain cleavage of the IgG.Final Fab concentration was 11 mg/ml in PBS. The digestion was performedover night at 37° C. using papain (Roche Diagnostics 108 014; 10 mg/ml)added to the antibody at a 1:100 ratio (w/w) and in the presence of 1 mMcysteine (added to the original IgG solution) The digestion was stoppedby addition of 50 μM of the specific papain inhibitor E64,(N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine, and thedigest was passed over a small protein A column (5 mL) in order toremove the Fc portion. The Fab was recovered in the flow-through,dialyzed against PBS, concentrated to its final concentration byultrafiltration and sterile filtered (0.22 μm).

Complex Formation, Crystallization and Structure Solution:

The FXI catalytic domain and the Fab were mixed at equimolar ratio andconcentrated to a final concentration of ca. 9 mg/ml.

The crystal used for data collection was obtained at 277K employingsitting drop vapor diffusion mixing 0.3 μL of reservoir solution (0.2 Mammonium-chloride, 20% PEG 3350), 0.2 μL Fab-FXI complex and 0.1 μLcrystal seeds from crystals obtained in a first round of crystalscreening.

For data collection crystals were directly flash frozen in liquidnitrogen. Data were collected at the Swiss Light Source beamline X10SAat a wavelength of 1.00002 Å using a Pilatus pixel detector (Dectris) at100K. Data processing and scaling was performed with XDS and XSCALE(Kabsch, W. (2010) Acta Cryst. D66, 125-132). The crystal diffracted toa resolution of 2.04 Å with unit cell dimensions of a=191.27, b=53.22,c=65.164 alpha=90.0, beta=94.56, gamma=90.0 (Space group C2) with onecopy of the complex per asymmetric unit.

The structure of the complex was solved by molecular replacement usingstructures of the FXI catalytic domain and a truncated Fab previouslysolved in-house as search models using PHASER (McCoy, A. J. et al.(2007) J. Appl. Cryst. 40, 658-674). Alternating cycles of refinementand rebuilding were performed using buster and coot rsp. (Bricogne, G.et al. (2010) BUSTER version 2.9. Cambridge, United Kingdom: GlobalPhasing Ltd.; Emsley, P. and Cowtan, K. (2004). Acta Crystallogr. D60,2126-2132). The data collection and refinement statistics are summarizedin Table 5.

TABLE 5 Data collection and refinement statistics Data collection Spacegroup C2 Cell dimensions a, b, c (Å) 191.27, 53.22, 65.16  α, β, γ (°)90, 94.56, 90 Resolution (Å) 64.96-2.04 (2.09-2.04) R_(sym) 0.099(0.571) // σ/ 10.8 (2.7) Completeness (%) 95.9 (98.4) Redundancy 3.2Refinement Resolution (Å) 64.96-2.03 No. reflections 40088R_(work)/R_(free) 0.217/0.282 No. atoms Protein 5071 Water 413 R.m.s.deviations Bond lengths (Å) 0.01 Bond angles (°) 1.19 *Values inparentheses are for highest-resolution shell.

Description of the Structure:

The structure reveals the binding epitope of the antibody NOV1401 bindsto the active site surface with the heavy chain CDR3 loop coveringportions of the S3, S2, S1-beta and S1′ subsites. The adjacent heavychain CDR1 and CDR2 loops induce conformational changes in the FXI 145-and 220-loops (chymotrypsin numbering). In addition, four N-terminal FXIresidues as well as residues surrounding Asp189 become disordered; bothare portions with key functions for catalytic activity of FXI. Theconformational change of the 145-loop leads to occlusion of the S1pocket by Arg144 and of the S2′ subsite by Tyr143. Antibody bindinghence leads to an inhibited conformation of FXI through multiplemechanisms.

The observed inhibited form shares features described for thefull-length zymogen form of FXI (PDB 2F83). The portions of the FXIcatalytic domain that have changed conformation or have becomedisordered as a result of antibody binding are disordered in the zymogenalso. This explains the strong binding of NOV1401 also to the zymogenform of FXI.

Our finding that NOV1401 does not inhibit FXI zymogen activation is inagreement with the distance of the binding epitope from the FXIa zymogenactivation cleavage site. NOV1401 binds both to FXI and FXIa. The X-raystructure of the Fab-FXI CD complex reveals a unique binding mode andmechanism of inhibition of FXIa. NOV1401 binds to the active site ofFXIa (FIG. 4) and induces conformational changes of the four N-terminalresidues and catalytic domain loops leading to an inactive conformation.This inactive conformation shares features with the inactive catalyticdomain structure in the zymogen (FIG. 5) providing an explanation howboth FXI and FXIa can be bound with high affinity by NOV1401. Forexample, three catalytic site loops (e.g., look 220, loop 188, and loop145) that are disordered in the zymogen structure are also disordered orshifted in the NOV1401 Fab-FXI CD complex structure, and an N-terminalsalt bridge observed in the active confirmation is absent in both thezymogen and NOV1401 Fab-FXI CD complex structures (Table 6). Hence,NOV1401 seems to induce conformational changes within the CD leading toan inactive, zymogen-like conformation.

TABLE 6 Structural features of FXIa CD, FXIa CD complexed with NOV1401and FXI-zymoqen (CD): NOV1401- FXIa FXI CD FXI CD complex zymogenSalt-bridge Ile16-Asp194 + − − Loop145 ordered shifted disorderedLoop188 ordered disordered disordered Loop220 ordered shifted disordered

Example 6 X-Ray Structure Based Epitope Mapping

Residues of FXI in contact with the Fab were analyzed using AREAIMOL(Briggs, P. J. (2000) CCP4 Newsletter No. 38), determining the residuesurface area difference when calculated without bound Fab and in complexwith the Fab, described as follows in Table 7 and Table 8a (Swissprotnumbering):

TABLE 7 FXI Epitope Epitope (Underlined: light chain & heavy chaincontacts): Light Chain Contacts Heavy Chain Contacts Pro410 Leu415Arg413 Cys416 His431 His431 Tyr434 Cys432 Gly435 Tyr434 Glu437 Tyr472Tyr472 Met474 Lys473 Ala475 Met474 Glu476 Glu476 Tyr521 Tyr521 Arg522Leu524 Lys523 Arg525 Leu524 Asp526 Arg525 His552 Asp526 Lys527 Arg548Ser575 Ser594 Trp595 Gly596 Glu597 Arg602 Glu603 Arg604

The FXI epitope is formed of the following residues:

Pro410, Arg413, Leu415, Cys416, His431, Cys432, Tyr434, Gly435, Glu437,Tyr472-Glu476, Tyr521-Lys527, Arg548, His552, Ser575, Ser594-Glu597,Arg602-Arg604.

TABLE 8a Residues of FXI In Contact with NOV1401 (Epitope) Residue AreaDifference PRO A 410 −11.0 ARG A 413 −36.3 LEU A 415 −3.6 CYS A 416 −2.1HIS A 431 −36.5 CYS A 432 −0.6 TYR A 434 −108.1 GLY A 435 −31.6 GLU A437 −3.7 TYR A 472 −13.0 LYS A 473 −40.1 MET A 474 −73.1 ALA A 475 −12.0GLU A 476 −40.4 TYR A 521 −18.5 ARG A 522 −46.6 LYS A 523 −74.7 LEU A524 −147.1 ARG A 525 −212.6 ASP A 526 −17.7 LYS A 527 −0.2 ARG A 548−11.6 HIS A 552 −4.0 SER A 575 −7.7 SER A 594 −8.7 TRP A 595 −20.9 GLY A596 −17.5 GLU A 597 −49.0 ARG A 602 −18.5 GLU A 603 −2.0 ARG A 604 −41.0

X-ray epitope mapped on the catalytic domain sequence (residues formingthe epitope bolded and underlined):

(SEQ ID NO: 51) 388 391 IVG GTASVRGEWP WQVTLHTTS P  TQ R H LCGGSI IGNQWILTAA  HC F YG V E SPK 441 ILRVYSGILN QSEIKEDTSF FGVQEIIIHD QYKMAE SGYD IALLKLETTV 491 NYTDSQRPIC LPSKGDRNVI YTDCWVTGWG  YRKLRDKIQN TLQKAKIPLV 541 TNEECQK R YR G H KITHKMIC AGYREGGKDA CKGD SGGPLS CKHNEVWHLV 591                                 625 GIT SWGE GCA QRER PGVYTN VVEYVDWILE KTQAV

Table 8b shows the residues of the antibody in contact with FXI(paratope).

TABLE 8b Residues of NOV1401 In Contact with FXI (Paratope). Residuedifference Area SER L 27 −1.80 GLY L 30 −5.00 SER L 31 −52.60 ASN L 32−21.00 ASP L 33 −22.00 TYR L 50 −36.00 LYS L 51 −54.20 TYR L 53 −41.40ASN L 54 −25.50 LYS L 67 −6.90 TRP L 92 −44.30 GLN L 94 −72.00 ARG L 95−5.70 PHE L 97 −54.70 ASP L 98 −2.70 VAL L 99 −0.10 PHE H 27 −2.00 THR H28 −20.50 SER H 30 −13.80 THR H 31 −78.90 ALA H 33 −10.80 TRP H 47−12.70 SER H 52 −2.20 TYR H 59 −62.00 TYR H 60 −0.80 GLU H 99 −1.70 SERH 101 −51.30 TYR H 102 −116.60 LEU H 103 −175.00 TYR H 104 −140.20 SER H105 −1.30 L, light chain; H, heavy chain

Example 7 Effect of FXI Antibody on FeCl₃-Induced Thrombosis in Mice

Mice deficient in FXI (FXI^(−/−) mice) on a C57Bl background were bredat Novartis (E. Hanover, N.J.) and used to assess the anti-thromboticefficacy of NOV1401. When reconstituted intravenously with human FXI(hFXI), these mice acquire a wild-type thrombophilic phenotype whenexposed to a thrombogenic stimulus. In the studies herein, thrombosiswas induced in the carotid artery by applying ferric chloride (FeCl₃) tothe surface of the artery.

NOV1401 was injected as a bolus through the jugular vein of anesthetizedmice 15 minutes prior to the induction of thrombosis. Doses of antibodyranged from 0.24 mg/kg-0.47 mg/kg. The FXI^(−/−) mice were reconstitutedwith human FXI by injecting 0.47 mg/kg human FXI via the jugular vein 10minutes prior to the FeCl₃ challenge. Two 1 mm×1.5 mm pieces of filterpaper saturated with 3.5% FeCl₃ were then applied to opposite sides ofthe carotid artery, in contact with its adventitial surface, and removed3 minutes afterward, followed by thorough washing with saline. Bloodflow through the carotid artery was measured with a Transonic flowprobe. Baseline blood flow was obtained for 5 minutes prior to FeCl₃application and then for 30 minutes after application of FeCl₃ (i.e.,during the thrombogenic period). At the end of the experiment blood wassampled from the vena cava into syringes containing 3.8% sodium citrate,plasma was prepared and subjected to an aPTT assay.

FIG. 1A shows the effect of NOV1401 on FeCl₃-induced thrombosis inFXI^(−/−) mice reconstituted with human FXI (humanized FXI mouse model).FIG. 1B shows the effect of NOV1401 on aPTT in the same mouse model.FIG. 1C shows aPTT prolongation in FXI^(−/−) mice in comparison towild-type mice.

NOV1401 fully inhibited FeCl₃-induced thrombus formation inhFXI-reconstituted FXI^(−/−) mice (FIG. 1A) starting at 0.24 mg/kg. Asteep dose response was observed, likely reflecting a stoichiometricall-or-none antithrombotic response. The aPTT was prolonged to 1.6 foldabove vehicle controls in the high dose group (FIG. 1 B), correspondingto the same level of prolongation by genetic depletion of FXI (FIG. 1C), i.e., maximal effect. These results show that NOV1401 hasanti-thrombotic activity in mouse FeCl₃ thrombosis model.

Example 8 Effects of FXI Antibody on Free FXI and aPTT in CynomolgusMonkeys

To evaluate the pharmacokinetic (PK) profile and pharmacological effectsof an anti-FXI/FXIa antibody, such as NOV1401, the antibody wasadministered via subcutaneous (s.c.) or intravenous (i.v.) injections tocynomolgus monkeys in a rising dose study.

The anticoagulant effect of NOV1401 was characterized in cynomolgusmonkeys by testing the antibody's ability to prolong aPTT and reducefree FXI (FXI_(f)) levels after a single intravenous (N=2) orsubcutaneous (N=2) dose of 3 mg/kg. A second dose of 10 mg/kg wasadministered to all animals followed by a third dose of 30 mg/kg todetermine if the effects observed at 3 mg/kg can be potentiated byhigher doses. These results show that NOV1401 has sustainedanticoagulant activity in cynomolgus monkeys. The pharmacodynamics (PD)of NOV1401, characterized by its anticoagulant effect as determined byaPTT and FXI_(f) levels, were then compared to the PK profile. Thecomparison indicates that there is a good PK/PD correlation.

Animals were dosed either i.v. (N=2) or s.c. (N=2) with NOV1401 on studyday 1 at 3 mg/kg, day 85 at 10 mg/kg and day 114 at 30 mg/kg. Bloodsamples were collected into sodium citrate coagulation tubes at 15 minand 2 hours post-dose for i.v. dosed animals, and for all animals atpretest, 6, 24, 48, 72 and 96 hours post-dose (days 1, 85 and 114) andat 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46, 50, 53, 57, 60, 64,66, 71, 75 and 78 days post-dose (day 1 only Blood was also collected ondays 92, 95, 99, 102, 107, 110, 121, 124, 128 and prior to dosing on day114. All blood samples were centrifuged; plasma samples were obtainedand frozen at approximately −70° C. or below.

Total NOV1401 plasma concentrations were measured by standard methodsfor human IgG detection by ELISA using a sandwich immunoassay with amouse anti-human-IgG monoclonal antibody as capture antibody and a goatanti-human-IgG with an HRP label as detection antibody.

For free FXI measurements in plasma samples that contain both FXI andNOV1401, unbound FXI was captured with immobilized NOV1401 and FXIalready complexed with NOV1401 was washed away. Plate-bound FXI was thendetected with a mouse Fc containing antibody 14E11, a monoclonalantibody that binds to the A2 domain of FXI and has been described inthe literature (Cheng, et al. Blood, 116:3981-3989, 2010). The very highaffinity of NOV1401 to both FXI and FXIa and the different binding sitesfor NOV1401 and the detection antibody 14E11 allowed an accuratedetermination of free FXI.

ELISA plates (384-Well LUMITRAC™ 600 HB) were coated with NOV1401 (5μg/mL in PBS) for binding of free FXI. After blocking (milk blocker: KPL#50-82-01, 1:20 dilution) and washing the plates with wash buffer (PBS;0.05% Tween 20), plasma samples diluted 1:40 in assay buffer (50 mMHEPES at pH 7.4, 125 mM NaCl, 5 mM CaCl₂, 5 mM EDTA and 0.05% (w/v)CHAPS) were incubated at RT for 30 min. and washed 3× with wash buffer.The detection antibody 14E11 was added at 1 μg/mL in dilution buffer(1.7 mM sodium phosphate monobasic, 8.1 mM sodium phosphate dibasicheptahydrate, 0.15 M sodium chloride, 0.7% Triton X-100, and 0.1% sodiumazide, pH 7) containing 0.7% casein. After washing the plates with washbuffer, a secondary detection antibody, peroxidase-labeled anti-mouseIgG (Sigma #A5278), was added at 0.5 μg/mL in dilution buffer containing0.4% casein. After washing the plates in wash buffer, 50 μL peroxidasechemiluminescent substrate solution (LumiGLO, KPL #54-61-01) was addedand the luminescence signal was read immediately on multi-modemicroplate reader (SPECTRAMAX M5E). The free FXI concentration in eachsample was determined using a standard curve generated with human FXI(zymogen) from Enzyme Research Laboratories (Catalog #HFXI 1111)starting from 100 nM FXI with a dilution factor of 2 and 22 dilutionsteps. The lower limit of quantification (LLOQ) was 0.24 nM FXI takinginto account the 1:40 dilution before measurement.

Plasma samples from all time points were subjected to aPTT analysis andaPTT results were compared to total plasma NOV1401 concentration andfree FXI levels. FIGS. 2A and 2B show changes of aPTT clotting times inrelationship to total plasma NOV1401 levels for i.v.- and s.c.-dosedanimals. FIGS. 3A and 3B show changes of aPTT clotting times inrelationship to free FXI levels for i.v.- and s.c.-dosed animals.

For i.v.-administered NOV1401, the highest plasma total NOV1401 levelswere observed at 15 min. post-dose (FIG. 2A). At this time the aPTT wasapproximately doubled versus baseline in both animals and remained atthis level for an average of 5-6 weeks. The mean aPTT prolongations from15 min. post-dose over the measurements preceding the decline towardbaseline were 2.0±0.02 times and 1.9±0.03 times for each animal.

By day 85, prior to administration of a second dose, aPTT had reachedbaseline levels and NOV1401 plasma concentrations had fallen below 10nM. A second dose of 10 mg/kg was administered on day 85 increasing theplasma concentration of total NOV1401 by about at least 3-fold andresulting in aPTT prolongation similar to what was observed after thefirst dose. A third dose of 30 mg/kg was administered on day 114 whileaPTT was still prolonged, and did not result in any significantadditional aPTT prolongation, despite another at least 3-fold increasein total NOV1401 plasma concentration (FIG. 2A). Therefore, NOV1401doses higher than 3 mg/kg achieved comparable aPTT prolongation as the 3mg/kg dose, and did not seem to increase the magnitude of aPTTprolongation. As expected, s.c. administration of NOV1401 resulted in aslower rise in aPTT than with i.v. administration, but the extent ofprolongation was comparable to that in the i.v. group (FIG. 2B). TheaPTT was prolonged versus baseline for an average of 5-6 weeks in thetwo animals. Mean aPTT fold prolongations were similar to those ofi.v.-treated animals: 2.0±0.03 and 1.8±0.02 from 6 hrs. post-dosethrough the measurements preceding the decline toward baseline. As inthe i.v, administering higher doses did not lead to higher aPTTresponses despite higher NOV1401 plasma exposures.

The results in FIGS. 2A-2B demonstrate that NOV1401 prolongs aPTT incynomolgus monkeys.

The mean baseline plasma FXI_(f) concentration was 10.9±0.3 nM in thei.v. group and fell rapidly (by 15 min.) upon injection of NOV1401 (FIG.3A). Plasma FXI_(f) levels remained low until total NOV1401 plasmalevels dropped to between 15 nM-25 nM (FIG. 2A, FIG. 3A). In the s.c.group, the mean baseline FXI_(f) concentration was 14.3±1.0 nM. FXI_(f)was sharply lower vs baseline by 6 hrs. post-treatment (FIG. 3B), andremained low until plasma NOV1401 levels declined to between 15 nM-25 nM(FIG. 2B, FIG. 3B). FXI_(f) dropped again sharply after the second doseat 10 mg/kg in all animals and remained low until the end of the study.The two higher doses did not further reduce FXI_(f) relative tobaseline.

In all treated animals, the drop and recovery of FXI_(f) levels weretemporally and inversely related to NOV1401-induced prolongation ofaPTT, confirming that NOV1401 inhibits the function of the intrinsiccoagulation pathway (prolongs aPTT) by lowering FXI_(f).

These results (e.g., FIGS. 3A and 3B) demonstrate that NOV1401 lowersplasma FXI_(f) levels in cynomolgus monkeys. In the cynomolgus monkeystudies, no evidence of excessive bleeding was observed at thevenipuncture sites or by gross observations at necropsy. Moreover,occult blood was not detected in stools throughout the study.

A sustained anticoagulant effect of NOV1401 was also observed in a13-week s.c./4-week i.v. repeat dose toxicity study in cynomolgusmonkeys. In this study, NOV1401 was administered weekly at doses of 10mg/kg (N=3, male and female combined) and 100 mg/kg (N=5, male andfemale combined) s.c. for 13 weeks (14 doses) or at 50 mg/kg (N=3, maleand female combined) i.v. for 4 weeks (5 doses). The control group (N=5,male and female combined) received vehicle for 13 and 4 weeks s.c. andi.v., respectively. FXI:C was assessed by measuring clotting time ofcynomolgus monkey plasma samples in the presence of human FXI deficientplasma (one-stage aPTT). aPTT and FXI:C were measured on study days 2,23, and 79 for the s.c. groups and on days 2 and 23 for the i.v. group.Across all animals and all treatment groups, an aPTT prolongation of2.1- to 3-fold was observed (FIG. 7A). The effect was sustainedthroughout the dosing phase of the study and no dose-dependency wasobserved similar to the observation in the previous rising dose study.FXI:C was reduced across animals and treatment groups by 88-95% andremained at these levels throughout the dosing phase of the study (FIG.7B). The effect on FXI:C was also dose-independent over these doses.

No evidence of macroscopic or microscopic indications of bleeding,including excessive bleeding, was observed at the venipuncture sites(including s.c. and i.v. injection and blood sampling sites) or by grossobservations at necropsy. Moreover, occult blood was not detected instools at the end of the study. In addition, no mortality occurred andthere were no test article-related effects on clinical signs, bodyweight, food consumption, ophthalmologic and electrocardiographicparameters, hematology, clinical chemistry, or urinalysis. No targetorgans of toxicity were identified.

Increased thyroid weights were observed in males at 100 mg/kg s.c.However, the toxicological significance of this finding is inconclusive,since there were no histologic correlates. There was large variabilityof thyroid weights amongst the animals, and the finding was present onlyin one sex. Microscopically, dose-dependent fibrosis at s.c. injectionsites in both sexes was observed at 10 and 100 mg/kg/week s.c. Thesefindings were not considered adverse.

No significant toxicity findings were observed in single rising dose orrepeat dose general toxicity studies in cynomolgus monkeys up to 13weeks. Therefore, the highest s.c. dose level administered in the13-week GLP toxicity study (100 mg/kg/week) was defined as the NOAEL.

Example 9 Pharmacokinetics in Cynomolgus Monkey—Single Dose

Cynomolgus monkeys (female, N=2) were administered a single 3 mg/kg doseof NOV1401 either i.v. or s.c. and observed until plasma FXI_(f)concentrations and aPTT returned to pre-dose values. The animals werethen administered a single 10 mg/kg dose of NOV1401 either i.v. or s.c.followed 2 weeks later by a 30 mg/kg dose either i.v. or s.c. andanother 2-week observation period. The PK of NOV1401 was assessed bymeasuring total NOV1401. The exposure to total NOV1401, as measured byeither the maximum observed total NOV1401 concentration (C_(max)) or thearea under the total NOV1401 concentration-time curve (AUC_(0-14d)), wascomparable between the individual animals in each group. Exposure(C_(max) or AUC_(0-14d)) was approximately dose-proportional for eachdosing route (Table 9). C_(max) was approximately 3-fold higher in thei.v. group than in the s.c. group. However, plasma total NOV1401concentrations were similar in both groups following the initialdistribution phase. The terminal elimination half-life (t₁₂) wasestimated for each animal using a two-compartment model followingadministration of the 3 mg/kg dose. The t₁₁₂ ranged from 14-15 days(N=2). The absolute bioavailability following s.c. injection ranged from61-66% (3 dose levels). Anti-NOV1401 antibodies were not detected aftereither i.v. or s.c. administration in any animals.

TABLE 9 Mean pharmacokinetic parameters following single (rising) doseadministration in female cynomolgus monkeys Dose t_(max) C_(max)AUC_(0-14 d) (mg/kg) Route (hr)* (μg/mL) (μg · d/mL) 3 i.v. 0.25 96.0544 3 s.c. 168 36.0 360 10 i.v. 0.25 325 1,810 10 s.c. 132 101 1,160 30i.v. 1.08 1,170 6,770 30 s.c. 132 344 4,140 *t_(max) is reported asmedian value.

Example 10 Toxicokinetics in Cynomolgus Monkey—Repeat Dose

Cynomolgus monkeys were administered weekly doses of 10 or 100 mg/kgNOV1401 s.c. for 13 weeks (14 doses) or doses of 50 mg/kg NOV1401 i.v.for 4 weeks (5 doses). Animals treated with NOV1401 were exposed toNOV1401 during the dosing phase of the study; no exposure was noted incontrol animals. No gender-related differences in exposure to plasmatotal NOV1401 were observed. The increase in exposure (both C_(max) andAUC_(0-7d)) was dose-proportional in both male and female animals (Table10). Anti-NOV1401 antibodies were detected in 5 of 6 animals at 10mg/kg/week s.c., in 1 of 10 animals at 100 mg/kg/week s.c., and in 1 of6 animals at 50 mg/kg/week i.v. Exposure to total NOV1401 was notcompromised in any of the s.c. dose groups. Only one anti-drug antibody(ADA)-positive animal had an AUC_(0-7d) on Study Day 22 that was lowerthan the other animals in the same group (50 mg/kg/week i.v.). There wasno impact on aPTT prolongation in this animal and no toxicity wasobserved.

TABLE 10 Mean toxicokinetic parameters for the penultimate dose (StudyDay 85 for the s.c. arms, Study Day 22 for the i.v. arm) of13-week/4-week GLP-compliant toxicity study in cynomolgus monkeys(male + female combined) Dose t_(max) C_(max) AUC_(0-14 d) (mg/kg/week)Route (hr)* (μg/mL) (μg · d/mL) 10 s.c. 24-120 719 3,100 100 s.c. 72-1205,630 23,400 50 i.v. 0.25-96   1,990 10,700 *t_(max) is reported as therange of values observed.

Example 11 Dose Escalation Study in Humans

Human studies are carried out to assess the safety and tolerability ofanti-FXI/FXIa antibodies, such as NOV1401, following single doseadministration in healthy subjects. A total of approximately 60 healthymale and post-menopausal/surgically sterile female subjects, between 18and 55 years of age, are entered into this study. Good health isdetermined by past medical history, physical examination, neurologicalexamination, vital signs, electrocardiogram (ECG), and laboratory testsat screening. Selected subjects weigh at least 50 kg, and have a bodymass index (BMI) within the range of 18-35 kg/m². BMI=body weight(kg)/[height (m)]².

Six s.c. dose levels of 5, 15, 50, 150, 300 and 600 mg are to be testedin a human study, provided that the predicted mean duration of aPTTprolongation ≥2-fold does not persist for 42 days at any tested dose.Two interim analyses (IA) are conducted to confirm dose selection forthe 2 highest dose levels. If the model-predicted mean duration of aPTTprolongation is 2-fold for longer than 42 days at the 300 mg or the 600mg dose, the dose can be lowered based, for example, based on modelsimulations, to ensure that the mean duration of aPTT prolongation doesnot exceed 2-fold for 42 days. Non-limiting exemplary dose adjustmentsmay involve lowering a dose using decrements of 10 mg, 20 mg, 30 mg, 40mg, or 50 mg.

The first three dose escalation steps occur at ≈1/2 log increments. Thelast 2 dose escalation steps are ≤2-fold increments to mitigate the riskof prolonged target saturation and extended aPTT prolongation.

The maximum duration of 2-fold aPTT prolongation for a certain number ofdays (e.g., 30 days, 35 days, 40 days, 42 days, etc.) with a therapytargeting FXI can be assessed based on genetic data showing mildbleeding phenotype in patients with severe FXI deficiency, data frompatients with FXI deficiency with acquired inhibitor, and also data fromhuman studies, for instance, FXI-ASO (see, e.g., Liu et al., (2011)“ISIS-FXIRx, a novel and specific antisense inhibitor of factor XI,caused significant reduction in FXI antigen and activity and increasedaPTT without causing bleeding in healthy volunteers.” Presented at the53rd American Society of Hematology annual meeting and exposition, SanDiego, Calif. Blood; 118: Abstract 209), where multiple doseadministration of FXI-ASO over 6 weeks resulted in a robust andsustained FXI depletion over >6 weeks (42 days) with no bleeding events.In certain embodiments, a model-based analysis predicts that maximumaPTT prolongation of ≈2.7-fold (relative to pre-dose) can be achievedtransiently at a 50 mg s.c. dose of NOV1401 (60-kg subject). In certainembodiments, higher doses are predicted to extend the duration of thismaximum aPTT prolongation of 2.7 fold.

Subjects are monitored throughout the study for safety parameters and/orend points, such as, physical exam, neurological exam, vital signs,electrocardiogram (ECG), safety laboratories, and adverse events (AEs)including serious AEs (SAEs) up until and including Day 106 post-dose.

The effect of anti-FXI/FXIa antibody (e.g., NOV1401) on aPTT is assessedbased on relative changes from baseline. Plasma concentrations of totalanti-FXI/FXIa antibody (e.g., NOV1401) are measured to assess the PK ofsingle doses in these subjects.

To assess immunogenicity (IG) of anti-FXI/FXIa antibodies (e.g.,NOV1401), screening and confirmation for ADA are conducted.

Free and total FXI and FXI coagulation activity (FXI:C) are measured toassess the effects of anti-FXI/FXIa antibodies (e.g., NOV1401) on targetengagement and target-related PD parameters.

D-dimer, prothrombin fragments 1.2 (F1.2) and prothrombin-antithrombincomplex (TAT) are assessed to determine the effects of anti-FXI/FXIaantibodies (e.g., NOV1401) on thrombogenesis parameters.

To study the effects of anti-FXI/FXIa antibodies (e.g., NOV1401) onother coagulation parameters, the following can be assessed: prothrombintime (PT), thrombin time (TT), and exploratory coagulation laboratoryparameters such as thrombin activatable fibrinolysis inhibitor,fibrinogen, tissue plasminogen activator (tPA) and TGA in the subjects.

Biomarkers studied may include, but are not be limited to: D-Dimer, FXIactivity, PT/INR, TT, F1.2, fibrinogen, TGA, TAFI activity, TAT, PAI-1antigen, TFPi activity, tPA activity, and vWF activity.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, publications, text books, and the like, and the references citedtherein, to the extent that they are not already, are herebyincorporated herein by reference in their entirety.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and examples detail certain preferred embodiments of theinvention and describe the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

1. (canceled)
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 5. (canceled) 6.(canceled)
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 10. (canceled) 11.(canceled)
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 15. (canceled)16. (canceled)
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 19. (canceled)
 20. Anisolated antibody or an antigen-binding fragment thereof whichspecifically binds to the catalytic domain of Factor XI (FXI) and/oractivated FXI (FXIa), wherein the antibody or fragment comprises a VHselected from the group consisting of SEQ ID NO: 29 or an amino acidsequence with 90% identity thereof; and a VL selected from the groupconsisting of SEQ ID NO: 19 and 39 or an amino acid sequence with 90%identity thereof.
 21. (canceled)
 22. (canceled)
 23. (canceled) 24.(canceled)
 25. (canceled)
 26. (canceled)
 27. The isolated antibody orfragment of claim 20, wherein the antibody or fragment comprises (i) aheavy chain variable region comprising CDR1 comprising the amino acidsequence of SEQ ID NO: 46; CDR2 comprising the amino acid sequence ofSEQ ID NO: 4; and CDR3 comprising the amino acid sequence of SEQ ID NO:5; and (ii) a light chain variable region comprising CDR1 comprising theamino acid sequence of SEQ ID NO: 33; CDR2 comprising the amino acidsequence of SEQ ID NO: 14; and CDR3 comprising the amino acid sequenceof SEQ ID NO:
 15. 28. The isolated antibody or fragment of claim 20,wherein the antibody or fragment comprises aa heavy chain variableregion comprising CDR1 selected from the group consisting of SEQ ID NO:3 and 23; CDR2 selected from the group consisting of SEQ ID NO: 4 and24; and CDR3 selected from the group consisting of 5 and 25; and (ii) alight chain variable region CDR1 selected from the group consisting ofSEQ ID NO: 13 and 33; CDR2 selected from the group consisting of SEQ IDNO: 14 and 34; and CDR3 selected from the group consisting of SEQ ID NO:15 and
 35. 29. The isolated antibody or fragment of claim 20, whereinthe antibody or fragment comprises a heavy chain variable regioncomprising CDR1 selected from the group consisting of SEQ ID NO: 6 and26; CDR2 selected from the group consisting of SEQ ID NO: 7 and 27; andCDR3 selected from the group consisting of 8 and 28; and (ii) a lightchain variable region comprising CDR1 selected from the group consistingof SEQ ID NO: 16 and 36; CDR2 selected from the group consisting of SEQID NO: 17 and 37; and CDR3 selected from the group consisting of SEQ IDNO: 18 and
 38. 30. (canceled)
 31. The isolated antibody or fragment ofclaim 20, wherein the antibody or fragment comprises a heavy chainvariable region CDR1 of SEQ ID NO: 23; a heavy chain variable regionCDR2 of SEQ ID NO: 24; a heavy chain variable region CDR3 of SEQ ID NO:25; a light chain variable region CDR1 of SEQ ID NO: 33; a light chainvariable region CDR2 of SEQ ID NO: 34; and a light chain variable regionCDR3 of SEQ ID NO:
 35. 32. (canceled)
 33. (canceled)
 34. Apharmaceutical composition comprising an antibody or fragment thereof ofclaim 20 and a pharmaceutically acceptable carrier.
 35. (canceled) 36.(canceled)
 37. The isolated antibody or fragment of claim 20, whereinthe antibody or fragment is selected from the group consisting ofNOV1090 and NOV1401.
 38. A method of treating a thromboembolic disordercomprising administering to a subject afflicted with a thromboembolicdisorder an effective amount of a pharmaceutical composition comprisingan antibody or fragment according to claim
 20. 39. The method of claim38, wherein the subject is afflicted with one or more of ischemic strokeassociated with atrial fibrillation and deep vein thrombosis. 40.(canceled)
 41. A method of treating a thromboembolic disorder comprisingadministering to a subject afflicted with a thromboembolic disorder aneffective amount of a pharmaceutical composition comprising an antibodyor fragment according to claim 20 in combination with statin therapies.42. (canceled)
 43. A nucleic acid coding for the antibody or fragmentthereof according to claim
 20. 44. A vector comprising the nucleic acidaccording to claim
 43. 45. A host cell comprising the vector of claim44.
 46. (canceled)
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 49. (canceled) 50.(canceled)
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 53. A method of treating orpreventing stroke in a patient with atrial fibrillation, comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition comprising the antibody or fragment of claim
 20. 54. Amethod of treating, managing or preventing deep vein thrombosis in apatient in need thereof, comprising administering to the subject aneffective amount of a pharmaceutical composition comprising the antibodyor fragment of claim
 20. 55. A method of treating, managing orpreventing venous thromboembolism in a patient in need thereof,comprising administering to the subject an effective amount of apharmaceutical composition comprising the antibody or fragment of claim20.
 56. A method of treating, managing or preventing thromboembolismover valvular mechanic or biologic prostheses, thrombosis of anyartificial surface in the arterial system, thrombosis on intra-arterialprosthesis or catheter, stent thrombosis, or thrombosis andthromboembolism associated with percutaneous coronary interventions, ina patient in need thereof, comprising administering to the subject aneffective amount of a pharmaceutical composition comprising the antibodyor fragment of claim
 20. 57. A method of managing bleeding in a patientbeing treated with an anti-FXI/FXIa antibody according to claim 20, saidmethod comprises temporarily reversing the anticoagulant effect of thean anti-FXI/FXIa antibody, wherein the step of reversing of theanticoagulant effect comprises (i) fluid replacement using colloids,crystalloids, human plasma or plasma proteins such as albumin; (ii)transfusion with packed red blood or whole blood; or (iii)administration of prohemostasis blood components selected from: freshfrozen plasma (FFP), prothrombin complex concentrates (PCC) andactivated PCC (APCC), and recombinant activated factor VII (rFVIIa).